Human disc tissue

ABSTRACT

This invention provides disc stem cells, processes for obtaining and culturing disc stem cells, and methods for repairing damaged or diseased disc tissue comprising the use of the disc stem cells of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application of PCT PatentApplication No. PCT/US2012/025066, filed Feb. 14, 2012, which claimspriority to U.S. Provisional Patent Application 61/442,315, filed Feb.14, 2011 and U.S. patent application Ser. No. 13/113,599, filed May 23,2011, which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

This invention provides disc stem cells, processes for obtaining andculturing disc stem cells, and methods for repairing damaged or diseaseddisc tissue comprising the use of the disc stem cells of the invention.

BACKGROUND OF THE INVENTION

Neck and lower back pain from degeneration of the spinal disc jointconstitutes a common and significant health and economic burden. Indeed,roughly 80% of adults will experience neck and lower back pain at somepoint in their lives due to intervertebral degeneration. Intervertebraldisc degeneration is a product of lifelong, slow, and chronicdegeneration that is synchronized with remodeling of the disc andneighboring bony structures. Musculoskeletal disorders of the spine andneck and lower back pain are the leading sources of disability in peopleless than 45 years of age, and lead to losses of over 90 billion dollarsa year in the U.S. They are the 2^(nd) most frequent reason to visit aphysician, the 5^(th) ranking cause of admission to the hospital, andthe 3^(rd) most common reason for surgical procedures.

Intervertebral degenerative disc disease contributes to structuralweakness of the outer capsule known as the annulus, which leads toherniation or protrusion of the disc into the spinal canal orneuroforamina where it impinges on traversing and exiting nerve roots.Intervertebral degenerative disc disease is characterized by aprogressive loss of proteoglycans and extracellular matrix in thenucleus pulposus, which causes loss of hydration and decreased jointmobility. The medical conditions associated with intervertebraldegenerative disc disease include disc herniation, radiculopathy,myelopathy, spinal stenosis, and all of these are closely associatedwith neck and lower back pain and extremity pain from affected nerveroots.

If a patient with neck and lower back pain fails to improve withconservative management, he is often referred for a surgicalintervention, of which the most common is a discectomy, with or withoutfusion. One alternative to discectomy is intervertebral artificial discinsertion, which is meant to provide a means of pain relief through astable motion-sparing reconstruction of the intervertebral segment. Thedrawbacks of this approach is the permanent disruption of the joint, anda high risk of chronic low grade arthritis in that joint secondary tothe natural biologic response of healthy or normal tissue to tissuedamage.

Another approach is a biological approach, in which non-disc cells,nucleus pulposus cells, or tissue engineered disc material is insertedinto the damaged NP to regenerate the matrix and restore the disc'sbiomechanical function. However, non-disc cells do not have theappropriate gene program required to survive in the harsh metabolicenvironment of the disc space, or produce the appropriate proteoglycanproducts, or have the intracellular biomechanical cytoskeleton andprotein machinery to withstand the constant and significant compressiveforces of the disc. Additionally, nucleus pulposus cells lack theability to divide more than a few times, and thus are not a sustainablecell source for long term regeneration. Finally, to date, a viablesource of tissue engineered disc material does not exist.

Tissue engineering approaches have not been successful to date, in partdue to limitations in knowledge and tissue biology relative to needs ofdisc tissue engineering and in part, to the harsh biomechanical andbiologic environment of the disc, which is inhospitable to most celltypes. Finally, in the absence of a disc tissue stem cell for tissueengineering, the use of other stem cell types such as mesenchymal stemcells and embryonic stem cells has been attempted, but they do notnaturally reprogram to functional disc cells successfully.

One branch of biologics uses stem cells such as mesenchymal stem cells,embryonic stem cells, and adipose stem cells in tissue engineering. Oneof the major obstacles in disc repair using stem cell therapy is theability to differentiate primary stem cell explants into the appropriatecell type and/or expand them in sufficient amounts for in vivotherapeutics. Very often, the process of reprogramming or “pushing” astem cell towards a given fate produces cells that may have some of theproperties of the targeted cell type, but are not adequately functional,or a cell population extremely limited in its ability to proliferatefurther once they are manipulated in vitro. The stem cells thus obtainedare not a viable product, because (a) they are too few; and (b) theylack the proper phenotype and/or function. Therefore, there is an urgentneed in the art for a technique that provides stem cells that may befurther expanded even after in vitro manipulation and maintain theproper phenotype and function. The present application answers thisneed.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method of amplifyingand enriching a disc stem cell population comprising the steps of: (a)culturing nucleus pulposus cells as an attached monolayer in a firstmedium comprising serum, basic fibroblast growth factor (bFGF),epidermal growth factor (EGF), and human neonatal foreskin fibroblast(NFF) cell supernatant on gelatin-coated tissue culture plates toconfluency; (b) isolating a single cell population of nucleus pulposuscells from said first medium; (c) culturing said nucleus pulposus cellsin suspension in a second medium comprising methylcellulose, serum,basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)on ultra-low binding culture plates until at least one of said cellsforms a sphere-like cluster, (d) isolating a population of disc stemcells from said second medium; (e) culturing said disc stem cells insuspension in a third medium comprising serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) on ultra-low bindingculture plates until at least some of said cells form an aggregatecluster, and (f) isolating a population of disc stem cells from saidthird medium, thereby amplifying and enriching a disc stem cellpopulation.

In another embodiment, the present invention provides a method ofamplifying a population of nucleus pulposus cells comprising the stepsof: (a) suspending isolated nucleus pulposus cells in a mediumcomprising approximately 14-15% serum, basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), and 30-40% human neonatalforeskin fibroblast (NFF) cell supernatant; and (b) plating thesuspension onto gelatin-coated tissue culture plates for 5-14 days,wherein said cells grow as an attached monolayer, thereby amplifying anucleus pulposus cell population.

In another embodiment, the present invention provides a method ofamplifying and enriching a disc stem cell population from nucleuspulposus cells comprising the steps of: (a) suspending a nucleuspulposus cell population comprising disc stem cells as single cells atlow density in a medium comprising 50% methylcellulose, 5% serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF); (b)distributing the suspension onto ultra-low binding culture plates; and(c) culturing the single cell suspension for 10-20 days, wherein anindividual disc stem cell grows into a sphere-like cell cluster in aclonal manner, thereby amplifying and enriching a disc stein cellpopulation.

In another embodiment, the present invention provides a method ofamplifying and enriching a disc stem cell population comprising thesteps of: (a) suspending a sphere-like cluster of disc stem cells or anisolated disc stem cell from said sphere-like cell cluster in a mediumcomprising 10% serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) and lacking methylcellulosc; (b)distributing the suspension onto ultra-low binding culture plates; (c)culturing said suspension for 8-16 days, wherein said cluster or cellgrows into a large aggregate cluster of heterogeneous morphology,thereby amplifying and enriching a disc stem cell population.

In another embodiment, the present invention provides an amplifiednucleus pulposus cell population obtained using a method comprising thesteps of: (a) suspending isolated nucleus pulposus cells in a mediumcomprising approximately 14-15% serum, basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), and 30-40% human neonatalforeskin fibroblast (NFF) cell supernatant; and (b) plating thesuspension onto gelatin-coated tissue culture plates for 5-14 days,wherein said cells grow as an attached monolayer, thereby producing anamplified nucleus pulposus cell population.

In another embodiment, the present invention provides a disc tissuederived clonal stem cell population obtained using a method comprisingthe steps of: (a) suspending a nucleus pulposus cell population in amedium comprising 50% methylcellulose, 5% serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF); (b) distributing thesuspension onto ultra-low binding culture plates; and (c) culturing saidsuspension for 10-20 days, wherein an individual disc stem cell growsinto a sphere-like cell cluster in a clonal manner, thereby producing adisc tissue derived clonal stem cell population.

In another embodiment, the present invention provides a disc tissuederived heterogeneous stem cell population obtained using a methodcomprising the steps of: (a) suspending a sphere-like cluster of discstem cells or an isolated disc stem cell from said sphere-like cellcluster in a medium comprising 10% serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) and lacking methylcellulose;(b) distributing the suspension onto ultra-low binding culture plates;(c) culturing said suspension for 8-16 days, wherein said cluster orcell grows into a large aggregate cluster of heterogeneous morphology,thereby producing a disc tissue derived heterogeneous stem cellpopulation.

In another embodiment, the present invention provides an amplified andenriched disc stein cell population obtained using a method comprisingthe steps of: (a) culturing nucleus pulposus cells as an attachedmonolayer in a first medium comprising serum, basic fibroblast growthfactor (bFGF), epidermal growth factor (EGF), and human neonatalforeskin fibroblast (NFF) cell supernatant on gelatin-coated tissueculture plates to confluency; (b) isolating a single cell population ofnucleus pulposus cells from said first medium; (c) culturing saidnucleus pulposus cells in suspension in a second medium comprisingmethylcellulose, serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) on ultra-low binding culture plates untilat least one of said cells forms a sphere-like cluster; (d) isolating apopulation of disc stem cells from said second medium; (e) culturingsaid disc stem cells in suspension in a third medium comprising serum,basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)on ultra-low binding culture plates until at least some of said cellsform an aggregate cluster; and (f) isolating a population of disc stemcells from said third medium, thereby producing an amplified andenriched disc stem cell population.

In another embodiment, the present invention provides a method oftreating a subject having a herniated disc, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) culturing nucleuspulposus cells as an attached monolayer in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates to confluency; (b) isolating asingle cell population of nucleus pulposus cells from said first medium;(c) culturing said nucleus pulposus cells in suspension in a secondmedium comprising methylcellulose, serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) on ultra-low binding cultureplates until at least one of said cells forms a sphere-like cluster; (d)isolating a population of disc stem cells from said second medium; (e)culturing said disc stem cells in suspension in a third mediumcomprising serum, basic fibroblast growth factor (bFGF), and epidermalgrowth factor (EGF) on ultra-low binding culture plates until at leastsome of said cells form an aggregate cluster; and (f) isolating apopulation of disc stem cells from said third medium, thereby producingsaid amplified and enriched disc stem cell population, and therebytreating said subject having a herniated disc.

In another embodiment, the present invention provides a method oftreating damage to or disease of the spinal joint of a subject,comprising the step of administering to said subject an amplified discstem cell population obtained using a method comprising the steps of:(a) culturing nucleus pulposus cells as an attached monolayer in a firstmedium comprising serum, basic fibroblast growth factor (bFGF),epidermal growth factor (EGF), and human neonatal foreskin fibroblast(NFF) cell supernatant on gelatin-coated tissue culture plates toconfluency; (b) isolating a single cell population of nucleus pulposuscells from said first medium; (c) culturing said nucleus pulposus cellsin suspension in a second medium comprising methylcellulose, serum,basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)on ultra-low binding culture plates until at least one of said cellsforms a sphere-like cluster, (d) isolating a population of disc stemcells from said second medium; (e) culturing said disc stem cells insuspension in a third medium comprising serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) on ultra-low bindingculture plates until at least some of said cells form an aggregatecluster; and (f) isolating a population of disc stem cells from saidthird medium, thereby producing said amplified and enriched disc stemcell population, and thereby treating damage to or disease of the spinaljoint of said subject.

In another embodiment, the present invention provides a method oftreating damage to or disease of a joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method comprising the steps of: (a)culturing nucleus pulposus cells as an attached monolayer in a firstmedium comprising serum, basic fibroblast growth factor (bFGF),epidermal growth factor (EGF), and human neonatal foreskin fibroblast(NFF) cell supernatant on gelatin-coated tissue culture plates toconfluency; (b) isolating a single cell population of nucleus pulposuscells from said first medium; (c) culturing said nucleus pulposus cellsin suspension in a second medium comprising methylcellulose, serum,basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)on ultra-low binding culture plates until at least one of said cellsforms a sphere-like cluster, (d) isolating a population of disc stemcells from said second medium; (e) culturing said disc stem cells insuspension in a third medium comprising serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) on ultra-low bindingculture plates until at least some of said cells form an aggregatecluster; and (f) isolating a population of disc stem cells from saidthird medium, thereby producing said amplified and enriched disc stemcell population, and thereby repairing said damaged or diseased disctissue.

In another embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of the spinal joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) culturing nucleuspulposus cells as an attached monolayer in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates to confluency; (b) isolating asingle cell population of nucleus pulposus cells from said first medium;(c) culturing said nucleus pulposus cells in suspension in a secondmedium comprising methylcellulose, serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) on ultra-low binding cultureplates until at least one of said cells forms a sphere-like cluster; (d)isolating a population of disc stem cells from said second medium; (e)culturing said disc stem cells in suspension in a third mediumcomprising serum, basic fibroblast growth factor (bFGF), and epidermalgrowth factor (EGF) on ultra-low binding culture plates until at leastsome of said cells form an aggregate cluster; and (f) isolating apopulation of disc stem cells from said third medium, thereby producingsaid amplified and enriched disc stein cell population, and therebypreventing, inhibiting, or decreasing the likelihood of damage to ordisease of the spinal joint of said subject.

In another embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of a cartilage-containing joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method comprising the steps of: (a)culturing nucleus pulposus cells as an attached monolayer in a firstmedium comprising serum, basic fibroblast growth factor (bFGF),epidermal growth factor (EGF), and human neonatal foreskin fibroblast(NFF) cell supernatant on gelatin-coated tissue culture plates toconfluency; (b) isolating a single cell population of nucleus pulposuscells from said first medium; (c) culturing said nucleus pulposus cellsin suspension in a second medium comprising methylcellulose, serum,basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)on ultra-low binding culture plates until at least one of said cellsforms a sphere-like cluster, (d) isolating a population of disc stemcells from said second medium; (e) culturing said disc stem cells insuspension in a third medium comprising serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) on ultra-low bindingculture plates until at least some of said cells form an aggregatecluster, and (f) isolating a population of disc stem cells from saidthird medium, thereby producing said amplified and enriched disc stemcell population, and thereby preventing, inhibiting, or decreasing thelikelihood of damage to or disease of the cartilage-containing joint ofsaid subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as the invention is particularly pointed outand distinctly claimed in the concluding portion of the specification.The invention, however, both as to organization and method of operation,together with objects, features, and advantages thereof, may best beunderstood by reference to the following detailed description when readwith the accompanying drawings in which:

FIG. 1. Schematic diagram of the Discotek Culture Platform. Preparationof a single cell suspension from a disc tissue specimen (referencenumerals 1-3); the Attachment Culture Method (reference numerals 4-7);the Discosphere Culture Method (reference numerals 8-13); and theCluster Culture Method (reference numerals 14-17). “Cryo” indicates thetiming of an optional cell cryopreservation step for each method.

FIG. 2. Schematic of the preparation of a single cell suspension from adisc tissue specimen.

FIG. 3. Preparation of a single cell suspension of human disc tissuesprior to plating cells in the culture method. A. Human disc tissues weredissected into smaller pieces, and washed thoroughly of blood and othertissue contaminants. The dissected fragments were placed in media withCollagenase II at 300 units/ml and incubated for 24 hours at 37° C. B. Alarge amount of debris was present post-digesion that made countingdifficult was removed with the washing step. A single cell suspensionfree of debris was the product of this method shown here at 10× (C.) and20× (D.) magnification.

FIG. 4. Preparation of a single cell suspension of porcine disc tissuesprior to plating cells in the culture method. A. Photograph ofpredissected and prewashed disc tissue fragments plated in a 10 cm dish.B. Photograph of predissected and postwashed disc tissue fragmentsplated in a T75 tissue culture flask for enzymatic digestion overnightfor 24 hours at 37° C. C. 20× photomicrograph of post-digested andwashed disc tissue cells in single cell suspension.

FIG. 5. Schematic of the Attachment Culture Method.

FIG. 6. The Attachment Culture Method at 24 hours postplating showingthe initial attachment of porcine disc cells. A.-C. Photomicrographs ofdisc tissues cells plated from the single cell suspension product of thetissue preparation method. The cells are plated on gelatin-coatedsurfaces in Discotek media. Photomicrographs of disc cells growingattached and in a monolayer at 4× (A), 10× (B), and 20× (C)magnification are shown.

FIG. 7. The Attachment Culture Method at 70% confluency showing thegrowth and development of porcine disc cells in the culture method.Photomicrographs of disc tissues cells plated from the single cellsuspension product of the tissue preparation method on gelatin coatedsurfaces in Discotek media and allowed to incubate for 3-5 days to reach70% confluency. Photomicrographs of disc cells growing attached as amonolayer at 4× (A), 10× (B), 20× (C), and 40× (D) magnification areshown.

FIG. 8. The Attachment Culture Method at confluency showing the growthand development of porcine disc cells in the culture method.Photomicrographs of disc tissues cells plated from the single cellsuspension product of the tissue preparation method on gelatin coatedsurfaces in Discotek media type 1 and allowed to incubate for 5-7 daysto reach confluency. Photomicrographs of disc cells growing attached asa monolayer at 10× (A), 20× (B), and 40× (C) magnification are shown.

FIG. 9. The Attachment Culture Method at confluency showing the growthand development of human disc cells in the culture method.Photomicrographs of disc tissues cells plated from the single cellsuspension product of the tissue preparation method on gelatin coatedsurfaces in Discotek media and allowed to incubate for 10-12 days toreach confluency. Photomicrographs of disc cells growing attached as amonolayer at 10× (A), 20× (B), and 40× (C) magnification are shown.

FIG. 10. Identification of notochordal cells in the Attachment CultureMethod after plating of the single cell suspension product of tissuepreparation. A. and B. 10× photomicrographs of a porcine single cellsuspension at the time of plating in the attachment culture before thecells have had a chance to attach (A), and after 24 hours of attachmentto gelatin on the plate's floor surface (B). C. 20× photomicrograph ofporcine disc cells in the attachment culture on the third day afterplating showing the persistence of notochordal cells (marked by a whitearrow).

FIG. 11. Schematic of the Discosphere Culture Method.

FIG. 12. The Discosphere Culture Method. A. 10× photomicrograph of humanstem cells growing as discospheres in the Discosphere Culture Method. B.A 20× photomicrograph of human discospheres growing in the DiscosphereCulture Method. C. and D. 40× photomicrographs of a single porcinediscosphere growing in the Discosphere Culture Method.

FIG. 13. Tissue engineering with Discospheres derived from theDiscosphere culture system. Discospheres are collected from the cultureand transferred to gelatin-coated coverslips and allowed to attach. A,C, and E: 20× photomicrographs taken at 24 hours, 48 hours, and 72 hourafter first plating the spheres. Figures B, D, and F are enlarged tofocus on the attached sphere of figures A, C, and E respectively, tobetter show cell activity at the tissue-surface interface and closelysurrounding areas. Note the significant morphologic changes in thesphere at 72 hours (E and F) and surrounding cells as compared toactivity at 24 hours (A and B).

FIG. 14. A second example of 2D tissue engineering with Discospheresattached to a coated culture surface. Photomicrographs taken at 48 hoursand 72 hours (shown at 20× magnification), and 96 hours and 120 hours(shown at 10× magnification) after first plating the spheres. Thisfigure demonstrates the morphology of cells as they differentiate intonucleus pulposus cells and migrate from the attached sphere cluster at48 hours. At 120 hours, the highly motile nature of the cells isapparent as the entire field is filled with migrating cells.

FIG. 15. Analysis of the expression of Vimentin in discospheres attachedto gelatin for eight hours. A. 100× light microscopy photomicrographdemonstrating the cellular architecture of the attached disc stem cellsphere from a human patient sample. B. 100× fluorescent confocalphotomicrograph demonstrating the immunohistochemical pattern ofVimentin in a sphere derived from a patient. C. 100× fluorescentconfocal photomicrograph demonstrating the immunohistochemicalexpression pattern of Vimentin of the attached sphere from a secondpatient sample. D. 100× fluorescent confocal photomicrographdemonstrating the immunohistochemical expression pattern of the negativecontrol for nonspecific antigen binding by the antibody.

FIG. 16. Analysis of the expression of Cytokeratin 8 (CK8) indiscospheres attached to gelatin for eight hours. A. 100× lightmicroscopy photograph demonstrating the cellular architecture of anattached disc stem cell sphere from a human patient sample. B. 100×fluorescent confocal photomicrograph demonstrating theimmunohistochemical expression pattern of CK8 in a sphere derived from apatient C. 100× fluorescent confocal photomicrograph demonstrating theimmunohistochemical pattern of CK8 of the attached sphere from a secondpatient sample. D. 100× fluorescent confocal photomicrographdemonstrating the immunohistochemical expression pattern of the negativecontrol for nonspecific antigen binding by the antibody.

FIG. 17. Analysis of the expression of Cytokeratin 8 (CK8) in disc cellsderived from Discosphere sphere culture, and attached to Gelatin forthree days. A. 40× light microscopy photograph demonstrating theattached cells on the gelatin-coated surface. B. 40× fluorescentphotomicrograph demonstrating the immunohistochemical expression patternof CK8 in the individual cells shown in A. C. 40× fluorescentphotomicrograph demonstrating the staining pattern of DAPI in theindividual cells shown in A and B.

FIG. 18. Schematic of the Cluster Culture Method.

FIG. 19. The Disc Cell Cluster Culture Method. A. 4× photomicrograph ofporcine disc clusters growing in the Cluster Culture Method. B. A 20×photomicrograph of porcine disc clusters growing in the Cluster CultureMethod. C. A 20× photomicrograph of porcine disc clusters growing in theculture method. D. 40× photomicrographs of a single porcine clustergrowing in the Cluster Culture Method.

FIG. 20. Analysis of the expression of CD133 in disc cells derived fromthe Cluster Culture Method and plated on gelatin for five hours. A-C.100× fluorescent photomicrographs of the expression of CD133 inindividual cells.

FIG. 21. 2D tissue engineering with Disc Cell Clusters attached to acoated culture surface. 20× photomicrographs taken at 48 hours, 72hours, 96 hours, and 120 hours after first plating the clusters.

FIG. 22. Analysis of the expression of Collagen-2 alpha and Vimentin indisc cells derived from the Cluster Culture Method and plated inchondrogenic differentiating conditions. A.-C. 100× fluorescent confocalphotomicrographs demonstrating the expression pattern of Collagen-2alpha in single disc cells differentiated from cluster cells of theCluster Cell Culture Method. D. and E. 100× fluorescent confocalphotomicrographs demonstrating the expression pattern of Vimentin insingle disc cells differentiated from cluster cells of the Cluster CellCulture Method. F. 100× fluorescent confocal photomicrographdemonstrating the immunohistochemical expression pattern of the negativecontrol for nonspecific antigen binding by the antibody.

FIG. 23. Schematic of the central role of OCT-4, NANOG, and SOX-2 inembryonic stem cell biology. OCT-4, in concert with NANOG and SOX-2, isa master transcriptional regulator of embryonic stem cell biology. Itspresence and activity in a stem cell indicates an early pluripotentstem-like state. These transcription factors regulate stem cell biologysuch as clonal division and the suppression of differentiation throughtheir target genes, the so-called NOS genes (NANOG, OCT-4, AND SOX-2).

FIG. 24. Experimental design for creating stably transfected pOCT-4-eGFPreporter expressing disc tissue cells. A. Schematic of the pOCT-4-eGFPreporter transgene (the full length OCT-4 promoter with all fourresponse elements cloned upstream of and regulating the expression ofgreen fluorescent protein (GFP). B. Schematic of the experimental designused for identifying and isolating cells that express GFP as driven bythe OCT-4 promoter. Briefly, disc stem cells were transfected with thepOCT-4-eGFP transgene. Stable transfectants were selected for theirresistance to the antibiotic G418 (via activation of the NEO resistancegene cassette also on the reporter plasmid).

FIG. 25. Demonstration of activation of the OCT-4 promoter-GFP reportertransgene in stably transfected disc tissue stem cells in the sphereculture.

FIG. 26. Organization of Spindle Shaped Nucleus Pulposus Cells in 2DTissue Engineering Assays. The left panels are light microscopyphotographs at 10× (top), 20× (middle), and 40× (bottom) magnifications.The right panels are gray scale mirror images with arrows placed withthe flow of disc cells along the surface of the plate.

FIG. 27 shows histology and proteoglycan assays of discospheres platedon gelatin coated coverslips. Discosphere remnants are surrounded byprogeny that express low levels of proteoglycans.

DETAILED DESCRIPTION OF THE INVENTION

The intervertebral disc consists of three main anatomic structures: thecartilaginous vertebral end plates, the annulus fibrosus (outer layer),and the nucleus pulposus (interior structure). In addition, thevertebral endplates are lined with a thin layer of hyaline cartilage.The annulus fibrosus (AF) consists of concentric lamellae of primarilycollagen I, and small amounts of several other collagens. The lamellaeare loosely bound to each other and contain fibroblast-like cells. Theinner portion of the annulus fibrosus comprises cells that resemblechondrocytes and merges with the nucleus pulposus at a transitional zonebetween the two types of tissues. The gelatinous interior nucleuspulposus (NP) is largely acellular and indeed is the largest avascularstructure in the body. It is a cartilage-like tissue with a molecularframework abundant in extracellular matrix that is made up of 50%proteoglycans (mainly aggrecan), 20% collagen II fibrils, and smallamounts of other collagens.

The spinal disc of all mammals originates from the notochord whichorganizes notochordal tissue and paraxial mesoderm into the fetal spine.

The inventors of the present application have discovered and developed adisc stem cell culture platform that enables isolation, culture, andexpansion of disc stem cells from adult disc tissue. Disc stem cellsprepared in this manner can be subsequently introduced into the discspace as a regenerative therapy after partial discectomy or discoplastyfor the treatment of severe degenerative disc disease and/or toreconstruct and regenerate defects in the joint that occur as sequalaeto discectomy.

The present invention provides compositions and methods of producing andusing said compositions that replace tissue degeneration or iatrogenicdisc defects due to surgery with tissue regeneration by combining tissueengineering with the proliferative and functional potential at thecellular level of adult disc stem cells. In particular, the inventors ofthe present application have developed a process to culture disc stemcells capable of forming discospheres and disc clusters, which may beused for in vitro and ex vivo research and development, and in vivotherapy of diseased or damaged disc tissue.

The present invention provides a method of isolating and culturing discstem cells, such that the disc stem cells maintain their functionalityand/or experience a gain in functionality and their ability to expand.The disc stem cells obtained by the methods of the present invention aremultipotent, as they can differentiate into different types of cells,each type expressing different biomarkers as described hereinbelow,having different morphologies, and giving rise to different phenotypes.Thus, in one embodiment, the disc stem cells of the present invention,when grown under the described conditions, mature into disc progenitorcells, which in turn develop into nucleus pulposus cells, which formdisc tissue upon maturation.

In one embodiment, the terms “differentiate” or “differentiation” referto the development of mature lineage specific cells with specializedstructure and function from immature unspecialized or less specializedprecursor cells, and includes the development of cells that possess thefunction of nucleus pulposus cells from precursor cells, disc stemcells, or disc progenitor cells.

In one embodiment, an isolated disc stem cell of the present inventionis derived from the nucleus pulposus tissue of a subject. The nucleuspulposus is a jelly-like substance at the center of the spinal discwhich comprises chondrocytes, collagen fibrils, and proteoglycans suchas hyaluronic acid and aggrecan which attract water.

In another embodiment, the present invention provides a population ofdisc stem cells that can form discospheres comprising disc stem cellsand early disc progenitor cells, which, in another embodiment may beused to produce nucleus pulposus cells.

In an additional embodiment, the present invention provides disc stemcells combined with biologically active factors useful for the treatmentin situ of degenerative disc disease, structural disc pathology, jointdestruction and annulus disruption, and regeneration of disc tissue.

In one embodiment, the isolated disc stem cell population of the presentinvention is a human disc stem cell population. In another embodiment,the isolated disc stem cell population of the present invention is anon-human disc stem cell population. In another embodiment, the isolateddisc stem cell population of the present invention is a mammalian discstem cell population. In another embodiment, the isolated disc stem cellpopulation of the present invention is a primate, feline, canine,bovine, ovine, porcine, equine, murine, or lapine (rabbit) disc stemcell population.

In another embodiment, the present invention provides a method ofcapturing, salvaging, stabilizing, and enriching rare stem cellfractions. In one embodiment, the methods of the present invention areparticularly useful when stem cells are present in small numbers, whichin one embodiment, is due to the nature of the tissue or species fromwhich the tissue is derived (e.g., human adult degenerative tissue andadult porcine tissue which are atypically acellular, etc.). In anotherembodiment, stem cells are present in small numbers, because thestarting tissue sample is small in weight, volume, or a combinationthereof.

In another embodiment, the present invention provides a method toenhance the stem cell potential of a stem cell population by drivingstem cells toward increased plasticity, immaturity, or a combinationthereof. In one embodiment, markers of immaturity include, inter alia,expression of embryonic transcription regulation factors such as OCT-4.

In one embodiment, disc stem cells of the present invention are able toboth proliferate under certain conditions that are described herein andto differentiate under certain conditions that are described herein andknown in the art.

In one embodiment, cells of the present invention are cultured in aculture vessel to purify or amplify a cell population. In oneembodiment, “culturing” is intended to refer to laboratory proceduresthat involve placing cells in culture medium f or an appropriate amountof time to allow stasis of the cells, or to allow the cells toproliferate, differentiate and/or secrete extracellular matrix.

In one embodiment, the present invention provides a method of producingan amplified disc stem cell population. In another embodiment, thepresent invention provides a method of producing an enriched disc stemcell population. In another embodiment, the present invention provides amethod of producing an amplified and enriched disc stem cell population.In one embodiment, the present invention provides a method of amplifyinga disc stem cell population.

In another embodiment, the present invention provides a method ofenriching a disc stem cell population. In another embodiment, thepresent invention provides a method of amplifying and enriching a discstem cell population.

In one embodiment, a disc stem cell population is amplified, enriched,or a combination thereof from nucleus pulposus cells, which in oneembodiment, are cells isolated from the nucleus pulposus. In oneembodiment, nucleus pulposus cells, as referred to herein, comprisemature nucleus pulposus cells, fibroblasts, nucleus pulposus stem cells,notochordal precursors, notochordal cells, or a combination thereof. Inanother embodiment, nucleus pulposus cells, as referred to herein,comprise mature nucleus pulposus cells, nucleus pulposus stem cells,notochordal precursors, and notochordal cells. In one embodiment, maturenucleus pulposus cells comprise differentiated nucleus pulposus cells,which, in one embodiment, express markers of differentiated nucleuspulposus cells as described and exemplified herein.

In one embodiment, “amplifying” a cell population refers to increasingthe number of cells in the population, regardless of cell type. Inanother embodiment, amplifying refers to equally increasing the numberof each cell type present in the population such that the percentage ofeach cell type in the population remains constant while the total numberof cells increases. In another embodiment, amplifying refers toincreasing the number of each cell type present in the population,wherein some cell types multiply at greater rates than others such thatthe percentage of each cell type in the population changes. In oneembodiment, amplifying refers to at least doubling the number of cellsin a population. In another embodiment, amplifying refers to at leasttripling the number of cells in a population. In another embodiment,amplifying refers to increasing the number of cells in a population byat least 50%. In another embodiment, amplifying refers to increasing thenumber of cells in a population by at least 75%. In another embodiment,amplifying refers to multiplying the number of cells in a population byat least 36. In another embodiment, amplifying refers to multiplying thenumber of cells in a population by at least 25. In another embodiment,amplifying refers to multiplying the number of cells in a population byat least 10. In another embodiment, amplifying refers to multiplying thenumber of cells in a population by at least 50. Thus, in one embodiment,an attachment culture amplifies stem cells. In another embodiment,sphere culture amplifies stem cells. In another embodiment, clusterculture amplifies stem cells.

In one embodiment, “enriching” a cell population refers to increasingthe percentage of a particular cell type in a population. In oneembodiment, enriching refers to increasing the percentage of a cell typein a population to 25%. In another embodiment, enriching refers toincreasing the percentage of a cell type in a population to 50%. Inanother embodiment, enriching refers to increasing the percentage of acell type in a population to 75%. In another embodiment, enrichingrefers to increasing the percentage of a cell type in a population to80%. In another embodiment, enriching refers to increasing thepercentage of a cell type in a population to 85%. In another embodiment,enriching refers to increasing the percentage of a cell type in apopulation to 90%. In another embodiment, enriching refers to increasingthe percentage of a cell type in a population to 95%. In anotherembodiment, enriching refers to increasing the percentage of a cell typein a population to 99%. Thus, in one embodiment, an attachment culturedoes not enrich for stem cells. In another embodiment, sphere cultureenriches for stem cells. In another embodiment, cluster culture enrichesfor stem cells.

In one embodiment, “culture vessel” refers to any container in whichcells may be cultured. Culture vessels include, but are not limited to,tissue culture flasks, 96 well plates, culture dishes, culture slides,and rotating wall vessels.

In another embodiment, the present invention provides a method to gentlyand safely derive single disc stein cells from disc tissue at higheryields than those produced with typical methods of tissue preparationcurrently available.

Accordingly, the present invention provides a method of isolating discstem cells from disc tissue obtained from a subject. In one embodiment,the subject is a mammal. In another embodiment, the subject is a human.In one embodiment, the method comprises mechanical disruption andenzymatic digestion for 24 hours of the tissue, washing to remove debrisand obtaining a cell suspension that comprises all disc tissue cells,including mature nucleus pulposus cells, disc stein cells, fibroblasts,notochordal cells, and several other cell types. The cell suspensioncontains all cell types in a manner which is roughly equivalent to thatof disc tissue, with disc stem cells representing less than 1% of thecell population, and nucleus pulposus cells representing 90% of the cellpopulation. The cells are then plated on gelatin-coated culture platesat 50,000 cells per cm² in a medium that contains modified humanneonatal foreskin fibroblast (NFF) conditioned media containingapproximately 15% serum, EGF, and basic fibroblast growth factor (bFGF).

In one embodiment, the disc stem cell population is 0.001 to 20% of thenucleus pulposus cell population. In one embodiment, the percentage ofdisc stem cells in a nucleus pulposus sample depends on the startingdisc tissue age, stage of development, disease status, species oforigin, or combination thereof. In one embodiment, all cell populationstypically present in the disc are represented in this culture, and allare increased in proportion during the culture. Thus, while theproportions of stem cells may remain the same after the culture method,the absolute numbers of each type of disc cell (most importantly discstem cells) are increased.

In one embodiment, disc tissue is procured from a human patient during asurgical procedure. In another embodiment, disc tissue is procured froman experimental animal model. In one embodiment, the tissue is processedby dissection and then enzymatically digested in media supplemented withCollagenase II, in one embodiment, at 200 units/ml, in one embodiment,for 24 hours, in one embodiment, at 37° C. for 24 hours. In anotherembodiment, tissue is enzymatically digested using hyaluronidase. Insome embodiments of the invention, non-nucleus pulposus or non-precursorcells are removed after hyaluronidase treatment using methods familiarto the skilled artisan, such as, for example, elutriation, whichinvolves differential centrifugation based upon the buoyant density ofthe cells, or centrifugation over a Percoll gradient. In one embodiment,cells from the digested tissue are put into a single cell suspension.

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained from a biopsy specimen of nucleus pulposus minced inpieces. In another embodiment, the pieces are 0.5-10 mm in size. Inanother embodiment, the pieces are 0.5-20 mm in size. In anotherembodiment, the pieces are 0.5-3 mm in size. In another embodiment, thepieces are 3-6 mm in size. In another embodiment, the pieces are 6-12 mmin size. In another embodiment, the pieces are 12-20 mm in size. Inanother embodiment, the pieces are 1-6 mm in size. In anotherembodiment, the pieces are 3-5 mm in size. In another embodiment, thepieces are 1-4 mm in size. In another embodiment, the pieces are 2-3 mmin size. In another embodiment, neonatal foreskin fibroblast tissue formaking neonatal foreskin fibroblast cell supernatant is minced inpieces. In one embodiment, neonatal foreskin fibroblast tissue is mincedinto pieces the size of which is described hereinabove.

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained from a biopsy specimen of nucleus pulposus by treatingnucleus pulposus tissue with a Collagenase solution (Example 1). Inanother embodiment, a heterogeneous population of nucleus pulposus cellsis obtained from a biopsy specimen of nucleus pulposus by treatingnucleus pulposus with a 0.1%-1% clostridial Collagenase (Worthington CLSII, 140 u/mg).

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained from a biopsy specimen of nucleus pulposus byaspiration of a disc of a subject. In another embodiment, aheterogeneous population of nucleus pulposus cells is obtained from abiopsy specimen of nucleus pulposus cells by aspiration through acannula, which in one embodiment, is part of a surgery, and in anotherembodiment, is percutaneous. In another embodiment, a heterogeneouspopulation of nucleus pulposus cells is obtained from a biopsy specimenof the nucleus pulposus tissue obtained from surgical resection, whichin one embodiment comprises surgery to gain exposure to the disc andthen resection of tissue.

In one embodiment, said subject is a patient. In another embodiment,said subject is a human. In another embodiment, said subject is ananimal. In one embodiment, said nucleus pulposus cells are from healthytissue. In another embodiment, said nucleus pulposus cells are fromdegenerated tissue.

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained from a biopsy specimen of nucleus pulposus byaspiration of a disc of a donor animal. In another embodiment, aheterogeneous population of nucleus pulposus cells is obtained from abiopsy specimen of nucleus pulposus by aspiration of a nucleus pulposusof a donor mammal. In another embodiment, a heterogeneous population ofnucleus pulposus cells is obtained from a biopsy specimen of nucleuspulposus by aspiration of a healthy disc of a patient.

In one embodiment, the nucleus pulposus cells are human nucleus pulposuscells. In another embodiment, the nucleus pulposus cells are mammaliannucleus pulposus cells. In another embodiment, the nucleus pulposuscells are isolated from degenerated disc tissue. In another embodiment,the nucleus pulposus cells are isolated from healthy disc tissue. In oneembodiment, the nucleus pulposus cells are isolated from fetal,neonatal, or young adult tissue.

Therefore, in one embodiment, the present invention provides a method ofamplifying a population of nucleus pulposus cells comprising the stepsof: (a) suspending isolated nucleus pulposus cells in a mediumcomprising serum, basic fibroblast growth factor (bFGF), epidermalgrowth factor (EGF), human neonatal foreskin fibroblast (NFF) cellsupernatant, or a combination thereof; and (b) plating the suspensiononto adherent tissue culture plates, thereby amplifying a population ofnucleus pulposus cells. In one embodiment, the adherent tissue cultureplate is a gelatin-coated tissue culture plate.

In another embodiment, the present invention provides a method ofamplifying a population of nucleus pulposus cells comprising the stepsof: (a) suspending isolated nucleus pulposus cells in a mediumcomprising approximately 14-15% serum, basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), and 30-40% human neonatalforeskin fibroblast (NFF) cell supernatant; and (b) plating thesuspension onto gelatin-coated tissue culture plates for 5-14 days,thereby amplifying a nucleus pulposus cell population. In oneembodiment, said cells grow as an attached monolayer on saidgelatin-coated tissue culture plates.

In one embodiment, said tissue culture plates are coated with 0.1%gelatin.

In one embodiment, the media comprising serum and NFF cell supernatantis referred to herein as “Discotek” medium. In another embodiment,Discotek medium comprises EGF and FGF2.

In one embodiment, a medium for use in the methods of the presentinvention comprises Dulbecco's Modified Eagle's Medium (DMEM). Inanother embodiment, a medium for use in the methods of the presentinvention comprises DMEM/F12. In another embodiment, a medium for use inthe methods of the present invention comprises DF10. In anotherembodiment, a medium for use in the methods of the present inventioncomprises Hamm's culture media. In another embodiment, a medium for usein the methods of the present invention comprises Hamm's/F12 culturemedia.

In another embodiment, a medium for the methods of the present inventionis supplemented with serum which in one embodiment is fetal bovine serum(FBS). In another embodiment, the serum is fetal calf serum (FCS). Inone embodiment, the concentration of serum is 8-20%. In anotherembodiment, the concentration of serum is 15%. In another embodiment,the concentration of serum is 5%. In another embodiment, theconcentration of serum is 6%. In another embodiment, the concentrationof serum is 10%. In another embodiment, the concentration of serum is14%. In another embodiment, the concentration of serum is 6-10%. Inanother embodiment, the concentration of serum is 5-10%. In anotherembodiment, the concentration of serum is 14.5%. In another embodiment,the concentration of serum is 13-16%. In another embodiment, theconcentration of serum is 11-20%. In another embodiment, theconcentration of serum is 14-15%. In another embodiment, theconcentration of serum is greater than 10%. In another embodiment, theconcentration is approximately the percentages described hereinabove. Inone embodiment, fetal bovine serum, NFF, fetal calf serum, DF10, or acombination thereof is a source of serum.

In another embodiment, the medium comprises NFF conditioned media. Inone embodiment, NFF are cultured in DF10 medium, and supernatant iscollected every 2 days, and frozen or used in a medium of the presentinvention as described herein. In one embodiment, the medium comprises33% media derived from cultures of primary NFFs. In another embodiment,the medium comprises 30-70% media derived from cultures of primary NFFs.In another embodiment, the medium comprises 30-35% media derived fromcultures of primary NFFs. In another embodiment, the medium comprises30-40% media derived from cultures of primary NFFs. In anotherembodiment, the medium comprises 25-50% media derived from cultures ofprimary NFFs.

In one embodiment, the nucleus pulposus cells demonstrate a notochordalcell morphology, a notochordal-like phenotype, or a combination thereof.In one embodiment, notochord cells with the distinctive morphologydescribed herein and in the literature is observed during the culture,but not when isolating cells from the culture. In one embodiment, themorphology of notochordal cells changes (becoming, in one embodiment,indistinct, difficult to identify, and similar to nucleus pulposuscells) under the conditions to which the cells are exposed. However,notochordal cells are still present, as is known in the art.

In one embodiment, said nucleus pulposus cells comprise chondrocyte-likecells. In another embodiment, said nucleus pulposus cells comprisenotochordal-like cells, which in one embodiment are larger thanchondrocyte-like cells, contain large vacuoles, and express proteins andother macromolecules important for disc cellular and structural biology.In another embodiment, said nucleus pulposus cells comprisenotochordal-like cells, which in one embodiment, have heterogeneousmorphology that vary according to culture conditions and can beidentified using molecular probes. In another embodiment, said nucleuspulposus cells comprise disc stem cells or nucleus pulposus stem cells.In one embodiment, depletion of notochordal cells correlates with theonset of disc degeneration. In one embodiment, a prolonged loss ofnotochord cells in the disc tissue correlates with an increased degreeof disc degeneration. In one embodiment, notochordal cells comprise discstem cells. In one embodiment, said nucleus pulposus cells comprise discstem cells, disc progenitor cells, mature disc cells, terminallydifferentiated disc cells, notochordal cells, or a combination thereof.

In one embodiment, nucleus pulposus cells derived directly from tissuepreparations are grown until confluent, at which time they are passagedinto the sphere culture. In one embodiment, cells are passaged when theyare 90% confluent. In another embodiment, cells are passaged when theyare 70% confluent. In another embodiment, cells are passaged when theyare 75% confluent. In another embodiment, cells are passaged when theyare 80% confluent. In another embodiment, cells are passaged when theyare 100% confluent.

In one embodiment, cells reach confluency in 3-5 days. In oneembodiment, cells reach confluency in 5-7 days. In one embodiment, cellsreach confluency in 4-7 days. In another embodiment, cells reachconfluency in 3-10 days. In another embodiment, cells reach confluencyin 10-12 days. In another embodiment, cells reach confluency in 7-14days. In another embodiment, cells reach confluency in 10-20 days. Inanother embodiment, cells reach confluency after 20 days. In anotherembodiment, cells reach confluency in 10-20 days. In another embodiment,cells reach confluency in 8-16 days.

In one embodiment, the nucleus pulposus cells reach 70% confluency aftera 3-5 day incubation. In another embodiment, nucleus pulposus cellsreach 100% confluency after a 5-7 day incubation. In another embodiment,nucleus pulposus cells reach 100% confluency after 7-14 days.

In one embodiment, cells are passaged by incubating them withCollagenase, which in one embodiment is Collagenase II. In oneembodiment, cells are incubated with Collagenase II overnight. Inanother embodiment, cells are incubated with Collagenase II for 1 hour.In another embodiment, cells are incubated with Collagenase II for 2hours. In another embodiment, cells are incubated with Collagenase IIfor 4 hours. In another embodiment, cells are incubated with CollagenaseII for 8 hours. In another embodiment, cells are incubated withCollagenase II for 12 hours. In another embodiment, cells are incubatedwith Collagenase II for 16 hours. In another embodiment, cells areincubated with Collagenase II for 24 hours. In one embodiment, theCollagenase is nucleolysin. In another embodiment, cells are incubatedwith chymopapain.

In one embodiment, methods of the present invention may comprise anincubation step wherein cells are incubated at 37° C. In anotherembodiment, cells are incubated at 35° C.-37° C. In another embodiment,cells are incubated at 33° C.-39° C. In another embodiment, cells areincubated at 37° C. In another embodiment, cells are incubated at 35° C.In another embodiment, cells are incubated at 36° C. In anotherembodiment, cells are incubated at 38° C. In another embodiment, cellsare incubated at 39° C. In another embodiment, cells are incubated at40° C. In another embodiment, cells are incubated at 41° C. In anotherembodiment, cells are incubated at 42° C.

In one embodiment, cells are incubated in an incubator maintaining 3-8%CO₂. In another embodiment, cells are incubated in an incubatormaintaining 4% CO₂. In another embodiment, cells are incubated in anincubator maintaining 5% CO₂. In another embodiment, cells are incubatedin an incubator maintaining 6% CO₂.

In one embodiment, cells are incubated under hypoxic conditions, whichin one embodiment comprises maintaining 2% O₂ in the incubator. Inanother embodiment, cells are incubated under normoxic conditions, whichin one embodiment comprises maintaining 20% O₂ in the incubator.

In one embodiment, cells are incubated in an incubator maintaining 60%humidity. In another embodiment, cells are incubated in an incubatormaintaining 70% humidity. In another embodiment, cells are incubated inan incubator maintaining 80% humidity. In another embodiment, cells areincubated in an incubator maintaining 90% humidity. In anotherembodiment, cells are incubated in an incubator maintaining 95%humidity.

In one embodiment, said nucleus pulposus cells are plated at highdensity for, in one embodiment, attachment culture. In one embodiment,said nucleus pulposus cells are plated at a cell surface density of50,000 cells/cm². In another embodiment, said nucleus pulposus cells areplated at a cell surface density of 40,000 cells/cm². In anotherembodiment, said nucleus pulposus cells are plated at a cell surfacedensity of 60,000 cells/cm². In another embodiment, said nucleuspulposus cells are plated at a cell surface density of 30,000 cells/cm².In another embodiment, said nucleus pulposus cells are plated at a cellsurface density of 70,000 cells/cm². In another embodiment, said nucleuspulposus cells are plated at a cell surface density of between30,000-70,000 cells/cm². In another embodiment, said nucleus pulposuscells are plated at a cell surface density of between 40,000-60,000cells/cm². In another embodiment, said nucleus pulposus cells are platedat a cell surface density of between 45,000-55,000 cells/cm². In anotherembodiment, said nucleus pulposus cells are plated at a cell surfacedensity of between 49,000-51,000 cells/cm².

In one embodiment, cells of the present invention are suspended at lowdensity for, in one embodiment, sphere culture and, in anotherembodiment, for cluster culture. In one embodiment, cells are suspendedat a final density of approximately 1×10⁴ cells/ml. In anotherembodiment, cells are suspended at a final density of approximately5×10⁴ cells/ml. In another embodiment, cells are suspended at a finaldensity of approximately 2×10⁴ cells/ml. In another embodiment, cellsare suspended at a final density of approximately 2×10⁵ cells/ml. Inanother embodiment, cells are suspended at a final density ofapproximately 1×10⁵ cells/ml. In another embodiment, cells are suspendedat a final density of approximately 1×10³ cells/ml. In anotherembodiment, cells are suspended at a final density of approximately5×10³ cells/ml. In another embodiment, cells are suspended at a finaldensity of approximately 5×10⁵ cells/ml. In another embodiment, cellsare suspended at a final density of approximately 1×10⁶ cells/ml. Inanother embodiment, cells are suspended at a final density ofapproximately 5×10⁵ cells/ml. In another embodiment, cells are suspendedat a final density of approximately 8×10⁴ cells/ml. In anotherembodiment, cells are suspended at a final density of approximately6×10⁴ cells/ml.

In another embodiment, the present invention further provides that discstem cells are grown in a medium comprising a compound that maintainscell juvenility or immaturity. In another embodiment, the presentinvention further provides that disc stem cells are grown in a mediumcomprising a compound which inhibits cell maturation. In anotherembodiment, the present invention further provides that disc stem cellsare grown in a medium comprising FGF which inhibits cell maturation. Inanother embodiment, the present invention further provides that discstem cells are grown in a medium comprising a compound that inhibitscell differentiation.

In one embodiment, any medium of the present invention is supplementedwith Fibroblast growth factor (FGF), which in one embodiment isfibroblast growth factor-2 (FGF-2), which in one embodiment, is alsoknown as basic Fibroblast Growth Factor (bFGF) or FGF-β. In anotherembodiment, the medium comprises 1-100 ng/ml FGF2. In anotherembodiment, the medium comprises 20-50 ng/ml FGF2. In anotherembodiment, the medium comprises 50-100 ng/ml FGF2. In anotherembodiment, the medium comprises 5-15 ng/ml FGF2. In another embodiment,the medium comprises 8-12 ng/ml FGF2. In another embodiment, the mediumcomprises 10 ng/ml FGF2.

In another embodiment, the present invention further provides that discstem cells are grown in a medium comprising a compound that promotesstem cell or precursor cell proliferation.

In one embodiment, a medium of the present invention is supplementedwith epidermal growth factor (EGF). In another embodiment, a medium ofthe present invention comprises 1-10 ng/ml EGF. In another embodiment,the medium comprises 1-100 ng/ml EGF. In another embodiment, the mediumcomprises 20-50 ng/ml EGF. In another embodiment, the medium comprises50-100 ng/ml EGF. In another embodiment, the medium comprises 5-15 ng/mlEGF. In another embodiment, the medium comprises 8-12 ng/ml EGF. Inanother embodiment, the medium comprises 10 ng/ml EGF.

In one embodiment, disc stein cells are grown in a medium of the presentinvention lacking heparin.

In another embodiment, the present invention further provides that discstem cells are grown in a medium comprising interleukin-2 (IL-2). Inanother embodiment, the present invention further provides that discstem cells are grown in a medium comprising interleukin-6 (IL-6). Inanother embodiment, the present invention further provides that discstem cells are grown in a medium comprising a stem cell factor (SCF).

In another embodiment, the present invention further provides that discstem cells are grown in a medium comprising transforming growth factor-β(TGF-β), which in one embodiment is TGFβ1 or TGFβ3. In anotherembodiment, the present invention further provides that disc stem cellsare grown in a medium comprising a TGF-β superfamily member. In anotherembodiment, the present invention further provides that disc stem cellsare grown in a medium comprising a BMP, which in one embodiment is BMP2,BMP4, or BMP7. In another embodiment, the present invention furtherprovides that disc stem cells are grown in a medium comprising a BMPsuperfamily member. In another embodiment, the present invention furtherprovides that disc stein cells are grown in a medium comprising an IL6cytokine family member, which in one embodiment, is leukemia inhibitoryfactor (LIF).

In some embodiments of the invention, a medium is supplemented withfibronectin at about 0.0001 to about 1 mg/ml. In some embodiments of theinvention, the medium is supplemented with TGF-β at about 10picograms/ml to about 10,000 picograms/ml, and more preferably at about100 picograms/ml to about 1000 picograms/ml; with PDGF at about 1.0ng/ml to about 10,000 ng/ml, and more preferably at about 10 ng/ml toabout 1000 ng/ml.

In some embodiments, biologically active molecules are included in amedium of the present invention. In one embodiment, the biologicallyactive molecules comprise growth factors, cytokines, antibiotics,proteins, anti-inflammatory agents, or a combination thereof. In oneembodiment, the biologically active molecules comprise TFG-beta, PDGF,EGF, FGF, IL-1 and IL-6.

In one embodiment, the medium is replenished every two days. In anotherembodiment, the medium is replenished every day. In another embodiment,the medium is replenished every three days. In another embodiment, themedium is replenished every four days. In another embodiment, the mediumis replenished 2-3 times per week.

In one embodiment, the growth and development of the cells are monitoredby the removal of an aliquot of the culture approximately every two daysand determining the DNA content of the cells. In one embodiment, thedegree of active proliferation by cells in culture is determined bydetermining DNA content. In one embodiment, the DNA content isdetermined by PI staining, FACS analysis, or a combination thereof. Inone embodiment, DNA content is reflective of the S phase (1-2× DNAcomplement) or G2 phase (2× complement) of cell cycling, or one of theother phases (1× DNA complement). In another embodiment, the growth anddevelopment of the cells are monitored by the removal of an aliquot ofthe culture and performing a cell count.

In one embodiment, any method of the present invention further comprisesthe step of cryopreserving the cells produced by one of the methodsdescribed herein. In one embodiment, two thirds of the total cellpopulation is cryopreserved. In one embodiment, cells are cryopreservedat 3M cells/ml, in one embodiment, in 1 ml aliquots. In one embodiment,cells are cryopreserved in Discotek media. In one embodiment,cryopreserved cells are used to generate stem cells, and, in anotherembodiment, cryopreserved cells are used at a later time forexperiments. In one embodiment, cells are cryopreserved at −20° C. Inanother embodiment, cells are cryopreserved at −70° C.-80° C. In oneembodiment, the cells may be cooled or frozen during storage to atemperature about or below 4° C. to about −196° C. In one embodiment,ultrarefined arabinogalactan is provided in the cryopreservation medium,optionally in combination with a second cryopreservation agent, such asdimethyl sulfoxide, in one embodiment, to protect the viability of cellsin the medium during the process of freezing, storage and thawing.

In one embodiment, the step of cryopreserving comprises suspending cellsin 10% DMSO plus 90% serum. In one embodiment, the step ofcryopreserving comprises aliquoting the mixture and placing the mixtureinto a cryopreservation vessel, which, in one embodiment is a structureto hold the vials, set within another vessel filled with isopropranol.In one embodiment, the step of cryopreserving comprises slowly freezingthe cells to a temperature of −80° C. In one embodiment, the step ofcryopreserving comprises transferring the cells to liquid nitrogen theday following the step of freezing the cells.

In one embodiment, any method of the present invention further comprisesthe step of preparing a single-cell suspension from the tissue or cellmonolayer or cellular aggregates produced by the method. In oneembodiment, cells are passaged by preparing a single cell suspension. Inone embodiment, enzymatic digestion is used to prepare the single cellsuspension.

In one embodiment, enzymatic digestion is used to create a single cellsuspension between each culturing step in the methods of the presentinvention. In one embodiment, tissue is procured, digested withCollagenase II for 24 hours, then the single cell preparation is platedin the attachment culture. Then, at confluency, the attachment cultureis prepared as a single cell suspension with Trypsin, and plated in thesphere culture. Then, at maturity, the sphere culture is digested withTrypsin, and plated as a single cell suspension into the clusterculture. In another embodiment, an alternative path is to transfer thesphere culture stem cell spheres directly to the cluster culture. Inanother embodiment, the sphere culture and the cluster culture can becycled within themselves up to three times, by creating a single cellsuspension with Trypsin, and replating in the same culture system. Inone embodiment, cells are expanded by around 1:3 ratios.

In one embodiment, the attachment culture method is only used once forexpansion of the cell populations. In one embodiment, if the cells arecycled repeatedly through the attachment culture method, thedifferentiation-driving aspects of the culture system (which, in oneembodiment, comprise attachment and serum) overwhelm the pro-stemaspects of the culture system, leading, in one embodiment, todifferentiation of the rare stem cell populations into other cell types.

The present inventors have made the unexpected discovery that disc stemcells may be successfully isolated from disc tissue and maintained inculture as disc stem cells when, in one embodiment, the cells are platedin low density in non-attachment culture plates in the presence ofmethylcellulose to prevent or minimize contact with other cells (e.g.stem cells, other cell types) and/or contact with any surface of theculture vessel. In one embodiment, these contacts may lead to or inducedifferentiation.

In one embodiment, cells are then suspended in media mixed 1:1 withmethylcellulose and culture media/supplemental reagents at low density(1×10⁴ cells/ml on tissue culture plates with ultra-low bindingsurfaces) such that the cells float and have no attachment to anysurface. This procedure, when combined with low density plating, allowsdisc stem cells to grow in isolation into monoclonally derived stem celland progenitor cell clusters. In fact, only stem cells can grow in theseconditions, whereas mature disc cells (nucleus pulposus cells) or latestage progenitors do not survive without attachment. The cells are thencultured from 12-16 days, while the media is changed every 2-3 days.

The method of the present invention allows disc stem cells to growsymmetrically or clonally to produce other disc stem cells.Specifically, the plated disc stem cells floating in media clonallydivide and replicate themselves at a rate of division that is muchhigher than the rate of symmetric division of stem cells in vivo in thenatural tissue environment. As the disc stem cells divide, they slowlygrow in size as a cluster of cells tightly attached to each otherthrough cell to cell contact, which, in one embodiment, occurs throughthe secondary secretion of extracellular matrix molecules (ECMs) (e.g.collagen, aggrecan, proteoglycans, etc.) that intercalate between thecells and around the aggregated cell body. ECMs typically have acement-like effect when secreted and remodeled in between two cellsalready in contact, or at least in close proximity.

These cell clusters are roughly spherical in shape, and, typically, theouter layers are more differentiated. In one embodiment, these sphericalcell clusters are referred to herein as “discospheres”. In oneembodiment, cells in a single discosphere that is in culture are derivedfrom the first isolated disc stem cell that was plated initially. Thisclonal growth, also known as symmetric growth, is, in one embodiment, acharacteristic feature of disc stem cells, and, in another embodiment,is one of the criteria that define a disc stem cell. In anotherembodiment, a discosphere of the present invention is the result of stemcell proliferation which gives rise to additional stem cells andprogenitor cells. In another embodiment, a discosphere is formed as aresult of disc stem cell proliferation.

In another embodiment, disc stem cells of the present inventionproliferate thus forming a discosphere. In another embodiment, adiscosphere of the present invention comprises nucleus pulposus stemcells and nucleus pulposus progenitor cells arranged in acircular-spherical structure. In another embodiment, a discosphere is aball of cells in which a single disc stem cell gives rise to clones ofitself (symmetric division) and to progenitor cells. In anotherembodiment, a discosphere of the present invention comprises freefloating nucleus pulposus stem cells and nucleus pulposus progenitorcells arranged in a circular-spherical structure. In another embodiment,a discosphere comprises nucleus pulposus cells that are attached to oneanother.

In one embodiment, the term “progenitor cells” refer to immaturestem-like cells with plastic potential and high proliferation rates,which can give rise to most if not all terminally differentiated tissuecells, but is not a disc stem cell.

In another embodiment, the present invention provides a method ofproducing an enriched disc stem cell population from nucleus pulposuscells comprising the steps of (a) suspending a nucleus pulposus cellpopulation in a medium comprising methylcellulose, fetal calf serum(FCS), basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), or a combination thereof; and (b) distributing the suspensiononto ultra-low binding culture plates, thereby producing an enricheddisc stem cell population.

In another embodiment, the present invention provides a method ofproducing an enriched disc stem cell population from nucleus pulposuscells comprising the steps of (a) suspending a nucleus pulposus cellpopulation in a medium comprising >10% methylcellulose, fetal calf serum(FCS), basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); and (b) distributing the suspension onto ultra-low bindingculture plates, wherein an individual disc stem cell grows into asphere-like cell cluster in a clonal manner, thereby producing anenriched disc stem cell population.

In another embodiment, the present invention provides a method ofproducing an enriched disc stem cell population from nucleus pulposuscells comprising the steps of: (a) suspending nucleus pulposus cellpopulation in a medium comprising >10% methylcellulose, 5-10% serum,basic fibroblast growth factor (bFGF), and epidermal growth factor(EGF); (b) distributing the suspension onto ultra-low binding cultureplates; and (c) growing said suspension for 10-20 days, wherein anindividual disc stem cell grows into a sphere-like cell cluster in aclonal manner, thereby producing an enriched disc stem cell population.

In another embodiment, the present invention provides a method ofproducing an enriched disc stem cell population from nucleus pulposuscells comprising the steps of: (a) suspending a nucleus pulposus cellpopulation comprising disc stem cells as single cells at low density ina medium comprising 50% methylcellulose, 5% serum, basic fibroblastgrowth factor (bFGF), and epidermal growth factor (EGF); (b)distributing the suspension onto ultra-low binding culture plates; and(c) growing said single cell suspension for 10-20 days, wherein anindividual disc stem cell grows into a sphere-like cell cluster in aclonal manner, thereby producing an enriched disc stem cell population.

In another embodiment, the method described directly hereinabove is amethod of producing a disc tissue-derived clonal stem cell population.

In one embodiment, a cell population comprises a multiplicity of cellsof heterogeneous cell types. In another embodiment, a cell populationcomprises a multiplicity of cells of a single cell type. In oneembodiment, a nucleus pulposus cell population comprises one or moredisc stem cell types.

In one embodiment, the step of suspending a cell population comprisessuspending individual cells in a liquid medium of varying degrees ofviscosity. In another embodiment, the step of suspending a cellpopulation comprises suspending single cells in a liquid medium ofvarying degrees of viscosity.

In one embodiment, the media comprises DMEM/F12 media. In anotherembodiment, the media comprises N2 Media. In another embodiment, themedia comprises DMEM/F12 and N2 Media.

In another embodiment, the present invention provides a media fordifferentiation of disc stem cells, which in one embodiment, comprisesone or more differentiation or pro-chondrogenic agents, which in oneembodiment, are BMP2, BMP7, TGFB1, TGFB3, dexamethasone, or acombination thereof.

In another embodiment, the present invention provides a compositioncomprising a discosphere. In another embodiment, a composition of thepresent invention comprises a single discosphere. In another embodiment,a composition of the present invention comprises at least 1×10²discospheres. In another embodiment, a composition of the presentinvention comprises at least 1×10³ discospheres. In another embodiment,a composition of the present invention comprises at least 1×10⁴discospheres. In another embodiment, a composition of the presentinvention comprises at least 1×10⁵ discospheres. In another embodiment,a composition of the present invention comprises at least 1×10⁶discospheres.

In another embodiment, the present invention provides that discospheresobtained by the methods of the present invention are further expanded.In another embodiment, the present invention provides that discospheresare dissociated by incubation at 37° C. in Trypsin. In anotherembodiment, the present invention provides that the dissociated cellsare expanded by replating the same into methylcellulose-based stem cellmedium and culture vessel as described hereinabove.

In one embodiment, the media for growing disc stem cells comprisesmethylcellulose. In one embodiment, the media for growing disc stemcells comprises >5% methylcellulose. In one embodiment, the media forgrowing disc stem cells comprises >3% methylcellulose. In oneembodiment, the media for growing disc stem cells comprises >2%methylcellulose. In another embodiment, the media for growing disc stemcells comprises 50% methylcellulose. In another embodiment, the mediafor growing disc stem cells comprises >10% methylcellulose. In anotherembodiment, the media for growing disc stem cells comprises >25%methylcellulose. In another embodiment, the media for growing disc stemcells comprises >40% methylcellulose. In another embodiment, the mediafor growing disc stem cells comprises 45-55% methylcellulosc. In anotherembodiment, the media for growing disc stem cells comprises 40-60%methylcellulose. In another embodiment, the media for growing disc stemcells comprises 30-70% methylcellulose.

In another embodiment, the method described hereinabove provides twodistinct sphere-shaped cellular products. In one embodiment, onesphere-shaped cellular product is a nucleus pulposus sphere, and in oneembodiment, a second sphere-shaped cellular product is a notochordalstem cell sphere.

In one embodiment, clonal refers to a group of cells that are derivedfrom a single cell and share many or all of its characteristics.

In one embodiment, the number of cells within a stem cell sphere and thesize of a stem cell sphere vary. In one embodiment, the number of cellsof a mature sphere is approximately 250 cells. In another embodiment,the number of cells of a mature sphere is approximately 100 cells. Inanother embodiment, the number of cells of a mature sphere isapproximately 500 cells. In another embodiment, the number of cells of amature sphere is approximately 100-500 cells. In another embodiment, thenumber of cells of a mature sphere is approximately 200-300 cells.

In one embodiment, the outer or peripheral layer of the sphere tends tobe somewhat more differentiated than the inner or core portion of thesphere, as is described herein (Example 4).

In another embodiment, the disc stem cells are plated on ultra lowattachment plates in the methods of the present invention. In anotherembodiment, the disc stem cells are plated on ultra low attachmentprecoated with an anti-adhesive substance plates in the methods of thepresent invention. In another embodiment, the anti-adhesive substance ispoly 2-hydroxyethyl methacrylate.

In one embodiment, disc stem cells produced by a method of the presentinvention express cytokeratin-8 (CK-8), CD-133, Nestin, and OCT-4.

In one embodiment, MMPs, ADAMTSs, and TIMPs are differentially expressedby chondrocytes and NP cells. In one embodiment, NP cells express higherlevels of MMP-2, MMP-14, ADAMTS-1,-2,-17 and TIMP-1 than chondrocytes.In another embodiment, NP cells express lower levels ofMMP-1,-3,-7,-8,-10,-11,-13,-16,-19,-20,-21,-23,-24,-28,ADAMTS-4,-5,-6,-14,-18,-19, and TIMP-3 than chondrocytes. In anotherembodiment, chondrocytes but not NP cells express MMP12 and MMP27.

In one embodiment, the method further comprises the step ofcryopreserving the sphere-like cell cluster (in one embodiment thediscosphere) or cells isolated from the sphere-like cell cluster.

When the discospheres reach a certain size, they need to be passaged tomaintain continued health and viability of the disc stem cellpopulation. Passaging involves collating all cultured discospheres intoa tube, washing and treating the cells with enzymes to break cell tocell contacts, and gentle pipetting of the cell pellet to break upclumps and create single cell suspensions enriched in disc stem cells(70-90% disc stem cells and early progenitors). The cells are counted atthis time to assess growth. The disc stem cells thus obtained may beanalyzed or cryopreserved directly, or converted back into single cellsuspensions and replated to continue their expansion in vitro. Thiscycle can continue indefinitely to expand the disc stem cell population,such that one cell can be expanded into 50 to 400 cells, in oneembodiment. The cells may be further expanded by a third passage forcryopreservation, cell and molecular assays or in vitro, and in vivotissue-engineering applications.

In another embodiment, the present invention provides a method ofamplifying an enriched disc stem cell population comprising the stepsof: (a) suspending a sphere-like cluster of disc stem cells or anisolated disc stem cell from said sphere-like cell cluster in a mediumcomprising fetal calf serum (FCS), basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), or a combination thereof andlacking methylcellulose; and (b) distributing the suspension ontoultra-low binding culture plates, thereby amplifying an enriched discstem cell population.

In another embodiment, the present invention provides a method ofamplifying and further enriching an enriched disc stem cell populationcomprising the steps of: (a) suspending a sphere-like cluster of discstem cells or an isolated disc stem cell from said sphere-like cellcluster in a medium comprising fetal calf serum (FCS), basic fibroblastgrowth factor (bFGF), and epidermal growth factor (EGF) and lackingmethylcellulose; and (b) distributing the suspension onto ultra-lowbinding culture plates, wherein said cluster or cell grows into a largeaggregate cellular cluster of heterogeneous morphology, therebyamplifying and further enriching an enriched disc stem cell population.

In another embodiment, the present invention provides a method ofamplifying and further enriching an enriched disc stem cell populationcomprising the steps of: (a) suspending a sphere-like cluster of discstem cells or an isolated disc stem cell from said sphere-like cellcluster in a medium comprising 10% serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) and lacking methylcellulose;(b) distributing the suspension onto ultra-low binding culture plates;(c) growing said suspension for 8-16 days, wherein said cluster or cellgrows into a large aggregate cluster of heterogeneous morphology,thereby amplifying and further enriching an enriched disc stem cellpopulation.

In another embodiment, the present invention provides a method ofamplifying and further enriching an enriched disc stem cell populationcomprising the steps of: (a) suspending a sphere-like cluster of discstem cells or an isolated disc stem cell from said sphere-like cellcluster in a medium comprising 10% serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF); (b) distributing thesuspension onto ultra-low binding culture plates; (c) growing saidsuspension for 8-16 days, wherein said cluster or cell grows into alarge aggregate cluster of heterogeneous morphology, thereby amplifyingand further enriching an enriched disc stem cell population.

In one embodiment, the medium as described hereinabove lacksmethylcellulose.

In another embodiment, the method described directly hereinabove is amethod of producing a disc tissue-derived heterogeneous stem cellpopulation.

In one embodiment, the media comprises DMEM/F12 media. In anotherembodiment, the media comprises N10 Media. In another embodiment, themedia comprises DMEM/F12 and N10 Media.

In one embodiment, the media further comprises putrescine, progesterone,sodium selenite, transferrin, insulin, or a combination thereof. Inanother embodiment, the media further comprises putrescine,progesterone, sodium selenite, transferrin, and insulin.

In one embodiment, N2 OR N10 media comprises DMEM F12 Media. In oneembodiment, N2 or N10 media comprises Sodium Selenite, which in oneembodiment is present in the media at a concentration of 30 μM. Inanother embodiment, the media comprises Sodium Selenite at aconcentration of 20-40 μM. In another embodiment, the media comprisesSodium Selenite at a concentration of 10-50 μM. In another embodiment,the media comprises Sodium Selenite at a concentration of 10-100 μM. Inanother embodiment, the media comprises Sodium Selenite at aconcentration of 25-35 μM.

In one embodiment, N2 OR N10 media comprises Insulin, which in oneembodiment is present in the media at a concentration of 5 μg/ml. Inanother embodiment, the media comprising disc stem cells furthercomprises 1-100 μg/ml insulin. In another embodiment, the mediacomprising disc stem cells further comprises 1-10 μg/ml insulin. Inanother embodiment, the media comprising disc stem cells furthercomprises 1-50 μg/ml insulin. In another embodiment, the mediacomprising disc stem cells further comprises 5-15 μg/ml insulin. Inanother embodiment, the media comprising disc stem cells furthercomprises 8-12 μg/ml insulin.

In one embodiment, N2 OR N10 media comprises Putrescine, which in oneembodiment is present in the media at a concentration of 100 μM. Inanother embodiment, the media comprises Putrescine at a concentration of1-200 μM. In another embodiment, the media comprises Putrescine at aconcentration of 1-500 μM. In another embodiment, the media comprisesPutrescine at a concentration of 50-150 μM. In another embodiment, themedia comprises Putrescine at a concentration of 100-200 μM. In anotherembodiment, the media comprises Putrescine at a concentration of 10-100μM. In another embodiment, the media comprises Putrescine at aconcentration of 75-125 μM.

In one embodiment, N2 OR N10 media comprises Progesterone, which in oneembodiment is present in the media at a concentration of 20 μM. Inanother embodiment, the media comprises Progesterone at a concentrationof 10-30 μM. In another embodiment, the media comprises Progesterone ata concentration of 1-50 μM. In another embodiment, the media comprisesProgesterone at a concentration of 15-25 μM. In another embodiment, themedia comprises Progesterone at a concentration of 10-50 μM. In anotherembodiment, the media comprises Progesterone at a concentration of10-100 μM.

In one embodiment, N2 OR N10 media comprises Transferrin, which in oneembodiment is present in the media at a concentration of 50 μg/ml. Inanother embodiment, the media comprising disc stein cells furthercomprises 1-400 μg/ml transferrin. In another embodiment, the mediacomprising disc stem cells further comprises 1-100 μg/ml transferrin. Inanother embodiment, the media comprising disc stem cells furthercomprises 20-150 μg/ml transferrin. In another embodiment, the mediacomprising disc stem cells further comprises 25-75 μg/ml transferrin. Inanother embodiment, the media comprising disc stem cells furthercomprises 40-60 μg/ml transferrin.

In one embodiment, N10 media further comprises serum, which in oneembodiment is fetal calf serum. In another embodiment, the serum isfetal bovine serum.

In one embodiment, said media comprises 10% serum. In one embodiment,said FGF-2 is present in the media at a concentration of 10 ng/ml. Inone embodiment, said EGF is present in the media at a concentration of10 ng/ml. In one embodiment, said suspension is distributed at a densityof 1×10⁴ cell/ml. In one embodiment, said suspension reaches confluencyafter approximately 10 days in culture.

In one embodiment, the method further comprises the step ofcryopreserving the large cluster of heterogeneous morphology or cellsisolated from the heterogeneous cluster.

In one embodiment, a method of the present invention further comprisesthe step of producing a single cell suspension from a sphere-like discstem cell cluster or a heterogeneous cluster using enzymatic digestionprior to the step wherein a single cell is suspended in a medium asdescribed herein.

In one embodiment, a method of the present invention further comprisesthe step of identifying disc stem cell markers and separating disc stemcells from disc progenitors and/or other cells present. According tothis aspect and in one embodiment, a method of the present inventionfurther comprises the step of sorting cells using biomarkers. In oneembodiment, FACS is used to sort cells. In another embodiment,magnetic-activated cell sorting (MACS) is used to sort cells, which inone embodiment, is a technique in which cells are sorted using a columnwith magnetic beads, wherein antibodies that bind the marker on stemcells are incubated with said stem cells, and then the solution isfiltered through the column where the antibody-biomarker-cell are pickedup by the beads, and the other cells flush through the column, and thenthe stem cells are eluted. In another embodiment, a reporter gene thatis genetically engineered to be regulated by the biomarker's promoter isused to sort cells. In one embodiment, an example of a reporter genethat is genetically engineered to be regulated by the biomarker'spromoter is OCT-4-GFP described hereinbelow. In one embodiment, CD133 isused for FACS and MACS. In one embodiment, OCT-4, CD133, and CK8promoters are engineered upstream of a reporter gene known in the artfor use in the method of cell sorting described hereinabove.

In one embodiment, the methods described hereinabove are combined in amethod of producing an enriched and amplified disc cell population.

According to this aspect and in one embodiment, the present inventionprovides a method of producing an amplified and enriched disc stem cellpopulation comprising the steps of: (a) growing isolated nucleuspulposus cells in a first culture comprising a medium comprising serumand human neonatal foreskin fibroblast (NFF) cell supernatant, andplated on gelatin-coated tissue culture plates to allow attachment andgrowth as a monolayer, (b) creating a single cell suspension of nucleuspulposus cells from said first culture; (c) growing disc stem cellsisolated from said first culture in a second culture comprising a mediumcomprising methylcellulose, and plating as a suspension on ultra-lowbinding culture plates, wherein individual disc stem cells grow intocellular clusters of a spherical shape; (d) isolating a population ofdisc stem cells from said second culture; and (e) growing disc stemcells or disc stem spheres isolated from said second culture in a thirdculture comprising a medium lacking methylcellulose, and plating thecells as a suspension on ultra-low binding culture plates, whereinindividual disc stem cells form aggregate clusters of heterogeneousmorphology; and (f) isolating a population of disc stem cells from saidthird culture, thereby producing an amplified and enriched disc cellpopulation. In one embodiment, said first culture is attachment culture.In one embodiment, said second culture is discosphere or sphere culture.In one embodiment, said third culture is cluster culture.

In one embodiment, the medium of step (a) comprises NFF supernatant. Inone embodiment, the medium of steps (a), (c), and (e) comprise basicfibroblast growth factor (bFGF), epidermal growth factor (EGF), or acombination thereof. In one embodiment, the medium of steps (a), (c),and (e) comprise serum, which in one embodiment, is fetal bovine serum(FBS).

In another embodiment, the present invention provides a method ofproducing an amplified and enriched disc stem cell population comprisingthe steps of: (a) growing nucleus pulposus cells as an attachedmonolayer in a first medium comprising serum, basic fibroblast growthfactor (bFGF), epidermal growth factor (EGF), and human neonatalforeskin fibroblast (NFF) cell supernatant on gelatin-coated tissueculture plates to confluency; (b) isolating a population of nucleuspulposus cells from said first medium; (c) growing said nucleus pulposuscells in a second medium comprising methylcellulose, serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF) onultra-low binding culture plates to confluency; (d) isolating apopulation of disc stem cells from said second medium; (e) growing saiddisc stem cells in suspension in a third medium comprising serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF) andlacking methylcellulose on ultra-low binding culture plates toconfluency; and (f) isolating a population of disc stem cells from saidthird medium, thereby producing an amplified and enriched disc stem cellpopulation.

In another embodiment, the present invention provides a method ofproducing an amplified and enriched disc stem cell population comprisingthe steps of: (a) growing nucleus pulposus cells as an attachedmonolayer in a first medium comprising serum, basic fibroblast growthfactor (bFGF), epidermal growth factor (EGF), and human neonatalforeskin fibroblast (NFF) cell supernatant on gelatin-coated tissueculture plates to confluency; (b) isolating a single cell population ofnucleus pulposus cells from said first medium; (c) growing said nucleuspulposus cells in suspension in a second medium comprisingmethylcellulose, serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) on ultra-low binding culture plates untilat least one of said cells forms a sphere-like cluster; (d) isolating apopulation of disc stem cells from said second medium; (e) growing saiddisc stem cells in suspension in a third medium comprising serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF) onultra-low binding culture plates until at least some of said cells forman aggregate cluster; and (f) isolating a population of disc stem cellsfrom said third medium, thereby producing an amplified and enriched discstem cell population.

In one embodiment, cells are grown in suspension in the second mediumuntil they reach maturity, which in one embodiment, comprises theformation of spheres comprised of individual cells that expanded in aclonal manner. In another embodiment, cells are grown in suspension inthe second medium until they reach a pre-determined size, or, in anotherembodiment, an appropriate size. In one embodiment, the pre-determinedsize is roughly 100 microns to 400 microns in diameter.

In another embodiment, cells are grown in suspension in the third mediumuntil they reach maturity, which in one embodiment, comprises theformation of cell aggregates, cell clusters, or aggregate cell clusters.In one embodiment, said aggregates, clusters, or aggregate clusters arenot geometric in shape but are still roughly spherical. In anotherembodiment, cells are grown in suspension in the third medium until theyreach a pre-determined size, or, in another embodiment, an appropriatesize. In one embodiment, the predetermined size is roughly 150 micronsto 600 microns in diameter.

In one embodiment, said first medium is attachment culture medium,which, in another embodiment, is called Discotek medium. In oneembodiment, said second medium is discosphere or sphere culture medium,which, in another embodiment, is called N5. In one embodiment, saidthird medium is cluster culture medium, which, in another embodiment, iscalled N10.

In another embodiment, the present invention provides a method ofproducing an amplified and enriched disc stem cell population comprisingthe steps of: (a) suspending isolated nucleus pulposus cells in a mediumcomprising approximately 15% serum, basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), and human neonatal foreskinfibroblast (NFF) cell supernatant; (b) plating the suspension ontogelatin-coated tissue culture plates wherein said cells grow as anattachment culture as a monolayer, thereby producing an amplifiednucleus pulposus cell population, (c) suspending said enriched disc stemcell population in a medium comprising 50% methylcellulose, fetal calfserum (FCS), basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); (d) distributing the suspension onto ultra-low bindingculture plates; wherein an individual disc stem cell grows into asphere-like cell cluster in a clonal manner, thereby producing anamplified and enriched disc stem cell population, (e) suspending saidamplified and enriched disc stem cell population in a medium comprisingfetal calf serum (FCS), basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) and lacking methylcellulose; and (f)distributing the suspension onto ultra-low binding culture plates,wherein said amplified disc stem cell population grows into a largecluster of heterogeneous morphology, thereby producing a furtheramplified and enriched disc stem cell population.

In another embodiment, the present invention provides a method ofproducing an amplified and enriched disc stem cell population comprisingthe steps of: (a) growing nucleus pulposus cells as an attachedmonolayer in a first medium comprising serum, basic fibroblast growthfactor (bFGF), epidermal growth factor (EGF), and human neonatalforeskin fibroblast (NFF) cell supernatant on gelatin-coated tissueculture plates to confluency; (b) isolating a population of nucleuspulposus cells from said first medium; (c) growing said nucleus pulposuscells in a second medium comprising methylcellulose, serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF) onultra-low binding culture plates to confluency; (d) isolating apopulation of disc stem cells from said second medium, thereby producingan amplified disc stem cell population.

In another embodiment, the present invention provides a method ofproducing an amplified and enriched disc stem cell population comprisingthe steps of: (a) suspending isolated nucleus pulposus cells in a mediumcomprising approximately 15% serum, basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), and human neonatal foreskinfibroblast (NFF) cell supernatant; (b) plating the suspension ontogelatin-coated tissue culture plates; thereby producing an amplifiednucleus pulposus cell population, (c) suspending said enriched disc stemcell population in a medium comprising >10% methylcellulose, fetal calfserum (FCS), basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); and (d) distributing the suspension onto ultra-low bindingculture plates; wherein an individual disc stem cell grows into asphere-like cell cluster in a clonal manner, thereby producing anamplified disc stem cell population.

In another embodiment, the present invention provides a method ofproducing an amplified and enriched disc stem cell population comprisingthe steps of: (a) growing nucleus pulposus cells as an attachedmonolayer in a first medium comprising serum, basic fibroblast growthfactor (bFGF), epidermal growth factor (EGF), and human neonatalforeskin fibroblast (NFF) cell supernatant on gelatin-coated tissueculture plates to confluency; (b) isolating a population of nucleuspulposus cells from said first medium; (c) growing said disc stem cellsin suspension in a second medium comprising serum, basic fibroblastgrowth factor (bFGF), and epidermal growth factor (EGF) and lackingmethylcellulose on ultra-low binding culture plates to confluency; and(d) isolating a population of disc stem cells from said second medium,thereby producing an amplified disc stem cell population.

In another embodiment, the present invention provides a method ofproducing an amplified and enriched disc stem cell population comprisingthe steps of: (a) suspending isolated nucleus pulposus cells in a mediumcomprising approximately 15% serum, basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), and human neonatal foreskinfibroblast (NFF) cell supernatant; (b) plating the suspension ontogelatin-coated tissue culture plates; thereby producing an amplifiednucleus pulposus cell population, (c) suspending said enriched disc stemcell population in a medium comprising fetal calf serum (FCS), basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF) andlacking methylcellulose; and (d) distributing the suspension ontoultra-low binding culture plates, wherein individual disc stem cellsform aggregate clusters, thereby producing an amplified disc stem cellpopulation.

In one embodiment, the methods of the present invention may be used toproduce a population of disc stem cells. In another embodiment, themethods of the present in invention may be used to produce a populationof non-disc stem cells, which in one embodiment, are adult or somaticstem cells, which in one embodiment, are tissue-specific stem cells. Inone embodiment, a tissue-specific stem cell is a stem cell from skin,muscle, intestine, or bone marrow. In another embodiment, atissue-specific stem cell is a neural stem cell, which in one embodimentis from the subventricular zone, the hippocampus, the cerebral cortex,or spinal cord. In another embodiment, a tissue-specific stem cell is astem cell from liver, heart, lung, pancreas, articular cartilage, bone,thymus, thyroid, or lymph node.

In one embodiment, an adult stem cell has the ability to self renew in aclonal manner, to divide asymmetrically to give rise to the differentcell lineages that are a part of the tissue from which the stem cell wasderived; to divide through multiple serial cell passages in vitro and invivo, or a combination thereof.

In another embodiment, the methods of the present in invention may beused to produce a population of endothelial stem cells, or dental pulpstem cells. In another embodiment, the methods of the present ininvention may be used to produce a population of fetal stem cells,embryonic stem cells, or cord blood stem cells. In such a case, a mediumfor nurturing the particular type of stein cell would be used, as isknown in the art together with the methods of the present invention. Inanother embodiment, the methods of the present in invention may be usedto produce a population of lung stem cells, heart stem cells, musclestem cells, brain stem cells, kidney stem cells, liver stem cells, dermastem cells, fat stem cells, or a combination thereof. In anotherembodiment, the methods of the present in invention may be used toproduce a population of Hematopoietic stem cells, mammary stem cells,mesenchymal stem cells, endothelial stem cells, neural stem cells,olfactory adult stem cells, neural crest stem cells, testicular cells,or a combination thereof.

In another embodiment, the invention provides a disc tissue derivedclonal disc stem cell population, and wherein said disc stem cells aremixture of functional disc stem cells and progenitors from two separategerm layers. In one embodiment, the present invention providesintervertebral disc tissue-derived mesodermal nucleus pulposus stemcells and progenitors that can be expanded for additional research,cryopreserved, and/or used directly for therapeutic applications. Inanother embodiment, the present invention provides intervertebral disctissue-derived neuroectodermal notochord stem cells and precursors, thatcan be expanded for additional research, cryopreserved, and useddirectly for therapeutic applications.

In one embodiment, the stem cell population may be exponentiallyexpanded by cycling through the Discosphere, Cluster Method stages, orboth the Discosphere and Cluster Method stages.

In one embodiment, the methods of the present invention amplify andenrich disc stem cells, while in another embodiment, they amplify andenrich disc progenitor cells.

In another embodiment, the present invention provides that plating aheterogeneous population of nucleus pulposus cells in a series of mediaas described herein comprising serum at low cell density results inisolation of nucleus pulposus stem cells. In another embodiment, thepresent invention provides that plating a heterogeneous population ofnucleus pulposus cells in a series of media as described hereincomprising serum at low cell density results in enriching a nucleuspulposus cell population for disc stem cells. In another embodiment, thepresent invention provides that plating a heterogeneous population ofnucleus pulposus cells at low cell density in a series of media asdescribed herein comprising serum, comprising a substance the interfereswith cell attachment results in the survival of nucleus pulposus stemcells. In another embodiment, the present invention provides thatplating a heterogeneous population of nucleus pulposus cells in a seriesof media as described herein, wherein one of the media comprises NFF andplating cells on gelatin coated plates at a high cell surface platingdensity, one comprises methylcellulose and plating at a low cell densityon ultra low binding tissue culture plates which interferes with cellattachment and N5 media, one comprises N10 media and plating on ultralow binding tissue culture plates, results in the survival of nucleuspulposus stem cells.

In another embodiment, the supplemented media of the present inventionenables only nucleus pulposus stem cells to grow. In another embodiment,the supplemented media of the present invention enable mostly nucleuspulposus stem cells to grow. In another embodiment, the supplementedmedia of the present invention preferentially encourages the growth ofnucleus pulposus stem cells.

In another embodiment, a medium for use in the present inventioncomprises an antibiotic supplemented to the media. In anotherembodiment, the antibiotic supplemented to the media ispenicillin-streptomycin. In another embodiment, a medium for use in thepresent invention comprises 1000-10000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 1000-3000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 3000-6000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 6000-10000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 3000-8000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 4000-6000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 5000 U/ml penicillin-streptomycinsupplemented to the media.

In another embodiment, the medium for growing disc stem cells, discprogenitor cells, or a combination thereof comprises a serum replacer,which in one embodiment is Invitrogen Knock-Out Serum Replacement,TM-235, CDM-HD, Omni Serum, or other serum replacers known in the art.In another embodiment, the methods of the present invention provide thatthe medium is free of FBS when growing disc stem cells, nucleus pulposusstem cells, progenitor cells, nucleus pulposus progenitor cells, or acombination thereof.

In another embodiment, a medium for use in the present inventioncomprises KO serum replacer supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.5-30%KO serum replacer supplemented to the media. In another embodiment, amedium for use in the present invention comprises 5-30% knockout (KO)serum replacer supplemented to the media. In another embodiment, amedium for use in the present invention comprises 3-5% KO serum replacersupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 5-15% KO serum replacer supplemented tothe media. In another embodiment, a medium for use in the presentinvention comprises 15-30% KO serum replacer supplemented to the media.In another embodiment, a medium for use in the present inventioncomprises 10-20% KO serum replacer supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 15-25%KO serum replacer supplemented to the media. In another embodiment, amedium for use in the present invention comprises 20% KO serum replacersupplemented to the media.

In another embodiment, a medium for use in the present inventioncomprises non-essential amino acids supplemented to the media. Inanother embodiment, a medium for use in the present invention comprises0.1-10% non-essential amino acids supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.1-1%non-essential amino acids supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 1-5%non-essential amino acids supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 5-10%non-essential amino acids supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 15-30%non-essential amino acids supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.5-1%non-essential amino acids supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.8-1.2%non-essential amino acids supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 1%non-essential amino acids supplemented to the media.

In another embodiment, a medium for use in the present inventioncomprises L-glutamine supplemented to the media. In another embodiment,a medium for use in the present invention comprises 0.1-10 mML-glutamine supplemented to the media. In another embodiment, a mediumfor use in the present invention comprises 0.1-5 mM L-glutaminesupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 5-10 mM L-glutamine supplemented to themedia. In another embodiment, a medium for use in the present inventioncomprises 5-8 mM L-glutamine supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.5-2.5mM L-glutamine supplemented to the media. In another embodiment, amedium for use in the present invention comprises 1.5-3 mM L-glutaminesupplemented to the media. In another embodiment, a medium for use inthe present invention comprises 0.5-1.5 mM L-glutamine supplemented tothe media. In another embodiment, a medium for use in the presentinvention comprises 0.8-1.2 mM L-glutamine supplemented to the media.

In another embodiment, a medium for use in the present inventioncomprises beta-mercaptoethanol supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.01-1mM beta-mercaptoethanol supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.01-0.5mM beta-mercaptoethanol supplemented to the media. In anotherembodiment, a medium for use in the present invention comprises 0.5-1 mMbeta-mercaptoethanol supplemented to the media. In another embodiment, amedium for use in the present invention comprises 0.5-0.8 mMbeta-mercaptoethanol supplemented to the media. In another embodiment, amedium for use in the present invention comprises 00.5-0.25 mMbeta-mercaptoethanol supplemented to the media. In another embodiment, amedium for use in the present invention comprises 0.15-0.3 mMbeta-mercaptoethanol supplemented to the media. In another embodiment, amedium for use in the present invention comprises 0.05-0.15 mMbeta-mercaptoethanol supplemented to the media. In another embodiment, amedium for use in the present invention comprises 0.08-0.12 mMbeta-mercaptoethanol supplemented to the media.

In another embodiment, the methods of the present invention provide thatan enriched nucleus pulposus stem cell population of the presentinvention comprises at least 60% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 70% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 80% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 85% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 90% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 95% nucleus pulposus stem cells.

In one embodiment, nucleus pulposus cells express genes that help themsurvive the hostile environment of the disc space, which in oneembodiment comprises high pressure, hypoglycemia, catabolic metabolism,acidic pH, low oxygen tension, and dynamic tissue biomechanics includingcontinuous compressive, sheer forces, or a combination thereof. In oneembodiment, nucleus pulposus cells express hypoxia induced Factor gene(HIF1a), glucose transporter 1 (GLUT1), or a combination thereof. In oneembodiment, cells expressing these proteins and other proteins of asimilar nature can survive and, in another embodiment, thrive in thedisc space. In another embodiment, nucleus pulposus cells expressproteoglycan products and intracellular biomechanical cytoskeleton andprotein machinery to withstand the constant and significant compressiveforces.

In one embodiment, the methods of the present invention provide purifieddisc stem cells. In one embodiment, “purified” refers to cells that aresubstantially free from other types of cells. In one embodiment,“substantially free from other types of cells” refers to cells that areat least 80% free from other types of cells, preferably at least 90%free from other types of cells, more preferably at least 95% free fromother types of cells, more preferably at least 98% free from other typesof cells, more preferably at least 99% free from other types of cells,and most preferably free from other types of cells.

In one embodiment, nucleus pulposus cells comprise inter alia precursorcells. In another embodiment, discospheres and/or clusters of thepresent invention comprise, inter alia, precursor cells. In oneembodiment, “precursor cells” refers to cells that, when cultured underappropriate conditions, develop into cells that possess the structureof, and function as, nucleus pulposus cells. Precursor cells include,but are not limited to, cells of the inner annulus fibrosus and nucleuspulposus.

In one embodiment, “nucleus pulposus cells” refers to cells that possessthe structure of, and function as, nucleus pulposus cells. Nucleuspulposus cells occupy the intervertebral disc, are relatively few innumber, and are surrounded by a hydrated (water containing)extracellular matrix that contains a high concentration of proteoglycan.The cells display prominent nuclei and secrete protcoglycans. Nucleuspulposus cells are present in the soft central portion of intervertebraldiscs and are mucoid in texture. Nucleus pulposus cells act as a cushionbetween the vertebrae by absorbing shock, and facilitate bending androtation of the vertebral column.

In one embodiment, nucleus pulposus cells comprise DNA, RNA, or proteinsthat serve as phenotypic markers and that allow nucleus pulposus cellsto be distinguished from other types of cells. Nucleus pulposusphenotypic markers include, but are not limited to, hypoxia inducingfactor-1.alpha. (HIF-1.alpha.), hypoxia inducing factor-1.beta.(HIF-1.beta.), glucose transporter-1 (GLUT-1), matrix metalloprotease-2(MMP-2), MMP-9, lactate dehydrogenase-A (LDH-A), thrombospondin-1(TSP-1), or a combination thereof. In another embodiment, nucleuspulposus phenotypic markers comprise phosphofructokinase-2 (PFK-2),GLUT-3, aggrecan, collagen type II, collagen type XI, Sox-9, or acombination thereof. In another embodiment, nucleus pulposus phenotypicmarkers comprise CD44 hyaluronan, receptor, beta-1 integrin subunit,endoglin/CD105, phosphorylated ERK, p38, or a combination thereof.

In one embodiment, a marker of a mature disc cell comprises collagen-2alpha, vimentin, aggrecan, or a combination thereof.

In another embodiment, nucleus pulposus cells may be identified bymorphological characteristics of nucleus pulposus cells. In oneembodiment, “morphological characteristics” is intended to refer to theform and structure of cells, and includes, but is not limited to, theshape and organization of cells, and the pattern formed by groups ofcells.

Notochordal cells can be identified through the recognition of theirdistinct cellular morphology, the presence of specific biomarkers, andother phenotypic characteristics found in vitro and in vivo. Notochordalcells tend to form clumps or aggregates of cells in most settings invitro or in vivo. In vivo the number of these clump are typically 5-10cells scattered in islands throughout the nucleus pulposus tissue. Invitro in cell culture after thorough digestion of the tissue,notochordal cells may be found in isolation and recognized by theirmorphology as being large vacuolated cells. In established cell cultureand stem cell culture, notochordal cells may be aggregates of 5-10 cellsup to 150-400 cells. Notochordal cells found in aggregate clumps orclusters typically secrete significant extracellular matrix which isreadily detected with light microscopy as a viscous rarefied gel-likecoat that surrounds and intercalates these multicelluar structures. Thegel like extracellular matrix is further detected in vivo and in vitroand characterized as such through histological stains that detectproteoglycans such as Toluidine and Alician Blue. Notochordal cells canbe identified by the expression of specific biomarkers, for example theexpression of CK8 and Vimentin, and their coexpression. Finally,especially in stem cell culture, notochordal clusters are atypicallyresistant to digestion with Trypsin, requiring prolonged incubationtimes for digestion, and the use of post-digestion techniques ofmechanical disruption of intracellular connections or bonding such astrituration.

Nucleus pulposus cells can also be identified, prior to isolation,during culture, and in post-culture assays, through the recognition ofphenotypic markers characteristic of nucleus pulposus cells. Phenotypicmarkers characteristic of nucleus pulposus cells have been ascertainedby identifying gene products whose expression is upregulated in responseto the biological program present in the nucleus pulposus. While nucleuspulposus cells share some of the characteristics of cartilage cells,they are embedded in a unique anatomical location that influences theirbiochemical and physiological characteristics. Nucleus pulposus tissueis avascular, and the absence of a vascular system imposes severerestrictions on the availability of oxygen, nutrients, and growthfactors to the cells. In addition, the osmotic pressure of theextracellular matrix is high, while the pH is low. To survive thesehostile conditions, nucleus pulposus cells have modified theirbiosynthetic pathways through the expression of a unique set of genes.The increased expression of certain proteins and genes in response tosevere oxygen and nutrient restriction provides a molecular profile thatcan be used to distinguish nucleus pulposus cells from cells of thesurrounding tissues.

Precursor cells can be identified using numerous methods familiar to oneof ordinary skill in the art. Once identified, and then isolated,precursor cells can be cultured under conditions effective to cause thecells to differentiate into nucleus pulposus cells.

In some embodiments of the invention, precursor cells can be identifiedby localizing proliferative centers in the disc unit. Proliferativecenters can be identified by various methods familiar to theart-skilled, including determination of the pattern ofbromodeoxy-uridine (BrdU) incorporation over time into the DNA of cellsof different regions of the disc, including the annulus fibrosus,vertebral end plates, and nucleus pulposus.

In one embodiment, a biomarker for a disc stem cell is OCT-4, nestin,CD133, CK8 or a combination thereof. In one embodiment, disc stem cellsproduced by a method of the present invention express CK8. In oneembodiment, disc stem cells produced by a method of the presentinvention express CD133. In another embodiment, stem cells produced by amethod of the present invention comprise activation of the OCT-4promoter in cells stably transfected with a reporter transgene constructconsisting of a full length human OCT-4 promoter with all fourregulatory elements, upstream of and directly regulating a greenfluorescent protein reporter gene. Activation of the OCT-4 promoter, asdescribed, indicates the expression of OCT-4 and implicates its activityin these stem cells to regulate it target genes (i.e., NOS genes).

In one embodiment, a biomarker for a mature nucleus pulposus cell isCollagen 2, Vimentin, Aggrecan, or a combination thereof. In oneembodiment, at least a portion of said stein cell population produced bya method of the present invention is capable of generating mature disccells that express the appropriate biomarkers, including in oneembodiment, Collagen II and Vimentin, after incubation with chondrogenicdifferentiating media. In another embodiment, at least a portion of saidstem cell population produced by a method of the present inventiongenerates mature disc cells that express the appropriate biomarkers,including Collagen II and Vimentin, after incubation with chondrogenicdifferentiating media. In one embodiment, chondrogenic differentiatingmedia comprises TGF-β1 and TGF-β3 (transforming growth factor beta), IGF(insulin-like growth factor), BMP2, BMP4, and BMP7 (bone morphogenicprotein), or a combination thereof. In one embodiment, chondrogenicdifferentiating media comprises dexamethasone, TGF-β1, TGFβ-3, BMP2,BMP4, and BMP7, or a combination thereof.

In one embodiment, “about” is intended to refer to plus or minus 10%.

In one embodiment, the present invention uses a sample as startingmaterial in the methods of the present invention. In one embodiment, theterm “sample” refers to biological material. The sample assayed by thepresent invention is not limited to any particular type. Samplesinclude, as non-limiting examples, single cells, multiple cells,tissues, biological fluids, biological molecules, or supernatants orextracts of any of the foregoing. Examples include tissue removed duringresection, blood, urine, lymph tissue, lymph fluid, cerebrospinal fluid,mucous, and stool samples. The sample used will vary based on the assayformat, the detection method and the nature of the tissues, cells orextracts to be assayed. Methods for preparing samples are well known inthe art and can be readily adapted in order to obtain a sample that iscompatible with the method utilized.

In one embodiment, the methods of the present invention further comprisedetecting biomarkers of a cell type. In one embodiment, the term“detecting” means to establish, discover, or ascertain evidence ofexpression of phenotypic markers of nucleus pulposus cells. Methods ofdetecting gene expression are well known to those of skill in the art.For example, methods of detecting nucleus pulposus markerpolynucleotides include, but are not limited of PCR, Northern blotting,Southern blotting, genomic approaches such as microarray, RNAprotection, and DNA hybridization (including in situ hybridization).Methods of detecting nucleus pulposus marker polypeptides include, butare not limited to, Western blotting, ELISA, enzyme activity assays,slot blotting, peptide mass fingerprinting, electrophoresis, fluorescentactivated cell sorting analysis (FACS), stable transfect ion of andassay of reporter transgenes, and immunohistochemistry. Other examplesof detection methods include, but are not limited to, histologic tissuestains and analysis, radioimmunoassay (RIA), chemiluminescenceimmunoassay, fluoroimmunoassay, time-resolved fluoroimmunoassay(TR-FIA), or immunochromatographic assay (ICA), all well known by thoseof skill in the art.

In one embodiment, the term “presence” refers to establishing that theitem in question is detected in levels greater than background.

In one embodiment, the phrase “evidence of expression of nucleuspulposus phenotypic markers” refers to any measurable indicia that anucleus pulposus phenotypic marker is expressed in the sample. Evidenceof nucleus pulposus phenotypic marker expression may be gained frommethods including, but not limited to, PCR, FISH, ELISA, IHC, FACS, orWestern blots.

In one embodiment, the amplified disc stem cells produced using themethods of the present invention are then exposed to differentiationmedium in order to produce nucleus pulposus cells. In one embodiment,differentiation medium comprises a medium supplemented with transforminggrowth factor β3 and β1 (TGF-β1; R&D Systems, MN), dexamethasone,ascorbate 2-phosphate, sodium pyruvate, proline, ITS-plus (CollaborativeBiomedical Products, Cambridge, Mass.), or a combination thereof. In oneembodiment, differentiation medium comprises DMEM supplemented with 10ng/mL TGFβ3 and β1, 100 nmol/L dexamethasone, 50 μg/mL ascorbate2-phosphate, 100 μg/mL sodium pyruvate, 40 μg/mL proline, and ITS-plus(Collaborative Biomedical Products, Cambridge, Mass.). In oneembodiment, controls for the experiment are disc stem cells maintainedmedia without any differentiating media supplements, or stem cellpromoting media.

In one embodiment, the methods of the present invention comprise thesteps described herein, in which additional method steps may also beincluded in the method. In another embodiment, the methods of thepresent invention consist of the steps described herein, in whichadditional method steps are not included in the method. In anotherembodiment, the methods of the present invention consist essentially ofthe steps described herein, in which additional method steps may also beincluded in the method but only additional steps that are not essentialto practicing the method.

In one embodiment, the present invention provides plating a suspensiononto adhesive-coated tissue culture plates. In one embodiment, saidadhesive is gelatin. In another embodiment, said adhesive is agar. Inanother embodiment, said adhesive is a mixture of agar and gelatin. Inanother embodiment, said adhesive is an extracellular matrix molecule orprotein, which in one embodiment is collagen or fibronectin. In anotherembodiment, said adhesive is poly-D-lysine. In another embodiment, saidadhesive is another adhesive known in the art.

In one embodiment, at least a portion of cells express a particularmarker. In one embodiment, at least a portion refers to at least 10%. Inanother embodiment, at least a portion refers to at least 20%. Inanother embodiment, at least a portion refers to at least 25%. Inanother embodiment, at least a portion refers to at least 30%. Inanother embodiment, at least a portion refers to at least 40%. Inanother embodiment, at least a portion refers to at least 50%. Inanother embodiment, at least a portion refers to at least 60%. Inanother embodiment, at least a portion refers to at least 70%. Inanother embodiment, at least a portion refers to at least 75%. Inanother embodiment, at least a portion refers to at least 80%. Inanother embodiment, at least a portion refers to at least 90%. Inanother embodiment, at least a portion refers to at least 95%.

In another embodiment, the present invention provides an amplified discstem cell population obtained using any of the methods describedhereinabove.

In another embodiment, the present invention provides an amplifiednucleus pulposus cell population produced using any of the methodsdescribed hereinabove. In another embodiment, the present inventionprovides an amplified nucleus pulposus cell population produced using amethod of amplifying a population of nucleus pulposus cells comprisingthe steps of: (a) suspending isolated nucleus pulposus cells in a mediumcomprising serum, basic fibroblast growth factor (bFGF), epidermalgrowth factor (EGF), and human neonatal foreskin fibroblast (NFF) cellsupernatant; and (b) plating the suspension onto gelatin-coated tissueculture plates, thereby producing an amplified nucleus pulposus cellpopulation. In one embodiment, these amplified nucleus pulposus cellscan be used in research and development into the characteristics ofmature vs. stem cells. In another embodiment, these amplified nucleuspulposus cells can be seeded with disc stem cells to improve theirgrowth potential and, in one embodiment, the final tissuenature/structure/biology of the disc stem cells.

In another embodiment, the present invention provides an enriched discstem cell population obtained using a method of producing an enricheddisc stem cell population from nucleus pulposus cells comprising thesteps of (a) suspending a disc tissue derived clonal stem cellpopulation in a medium comprising >10% methylcellulose, serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF); and(b) distributing the suspension onto ultra-low binding culture plates,wherein an individual disc stem cell grows into a sphere-like cellcluster in a clonal manner, thereby producing an enriched disc stem cellpopulation.

In one embodiment, the serum is fetal calf serum. In one embodiment,said serum is present in said media at 5%.

In another embodiment, the present invention provides a disc tissuederived clonal stem cell population obtained using a method of producingan enriched disc stem cell population from nucleus pulposus cellscomprising the steps of (a) suspending a disc tissue derived cellpopulation in a medium comprising >10% methylcellulose, serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF); and(b) distributing the suspension onto ultra-low binding culture plates,wherein individual disc stem cells grow in a clonal manner intomulticellular clusters of spherical shapes, thereby producing a disctissue derived clonal stem cell population.

In another embodiment, the present invention provides a disc tissuederived clonal stem cell population obtained using a method comprisingthe steps of: (a) suspending a nucleus pulposus cell populationcomprising disc stem cells as single cells at low density in a mediumcomprising 50% methylcellulose, 5% serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF); (b) distributing thesuspension onto ultra-low binding culture plates; and (c) growing saidsingle cell suspension for 10-20 days, wherein an individual disc stemcell grows into a sphere-like cell cluster in a clonal manner, therebyproducing an enriched disc stem cell population.

In one embodiment, a sphere-like cell cluster arises from thediscosphere or sphere culture conditions as described hereinabove. Inone embodiment, conditions (low cell density, methylcellulose, lowattachment plates) lead to conditions where cells are unable to attachto each other or to the plates. In one embodiment, these conditions leadto the survival of only stem cells, which grow in a multicellularstructure originating from a single cell so that the progeny areclonally derived.

In one embodiment, the disc tissue derived clonal disc stem cellpopulation may be cryopreserved, or used directly for research,experimental therapeutics, definitive therapeutics, or a combinationthereof.

In another embodiment, the present invention provides a disc tissuederived heterogeneous stem cell population obtained using a method ofamplifying a disc stem cell population comprising the steps of: (a)suspending a sphere-like cluster of disc stem cells or an isolated discstem cell from said sphere-like cell cluster in a medium comprisingserum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF) and lacking methylcellulose; and (b) distributing thesuspension onto ultra-low binding culture plates, wherein said clusteror cell grows into a large aggregate cluster of heterogeneousmorphology, thereby producing a disc tissue derived heterogeneous stemcell population.

In another embodiment, the present invention provides an amplified discstem cell population obtained using a method of amplifying a disc stemcell population comprising the steps of: (a) suspending a sphere-likecluster of disc stem cells or an isolated disc stem cell from saidsphere-like cell cluster in a medium comprising serum, basic fibroblastgrowth factor (bFGF), and epidermal growth factor (EGF) and lackingmethylcellulose; and (b) distributing the suspension onto ultra-lowbinding culture plates, wherein said cluster or cell grows into a largeaggregate cluster of heterogeneous morphology, thereby producing anamplified disc stem cell population.

In another embodiment, the present invention provides a disc tissuederived heterogeneous stem cell population obtained using a methodcomprising the steps of: (a) suspending a sphere-like cluster of discstem cells or an isolated disc stem cell from said sphere-like cellcluster in a medium comprising 10% serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF); (b) distributing thesuspension onto ultra-low binding culture plates; (c) growing saidsuspension for 8-16 days, wherein said cluster or cell grows into alarge aggregate cluster of heterogeneous morphology, thereby amplifyingand further enriching an enriched disc stem cell population.

In one embodiment, a heterogeneous stem cell population arises from theculture conditions of the cluster culture described hereinabove. In oneembodiment, a heterogeneous stem cell population comprises cells from asingle cluster, single cells, intermediate aggregates, or a combinationthereof. In one embodiment, disc stem cells cultured in suspension inlow density in a medium lacking methylcellulose stick to each other andto large clusters, resulting in a heterogeneous population of cells. Inone embodiment, the heterogeneous stem cell population comprisesclusters comprising a single disc stem cell clonally reproduced incontact with single stem cells or one or more additional clonallyreproduced disc stem cells. In another embodiment, the cell-cell contactinduces limited differentiation so that the heterogeneous stem cellpopulation comprises disc stem cells, early progenitor cells, and asmall percentage of more mature cells on the edges where most of thecell-cell contact occurs. In one embodiment, cultures of the presentinvention, and specifically cluster culture, are incubated with gentleagitation, as is known in the art.

In another embodiment, the present invention provides an amplified andenriched disc stem cell population obtained using any of the methods ofproducing an amplified and enriched disc stem cell population describedherein. In one embodiment, the present invention provides an amplifiedand enriched disc stem cell population obtained using a method ofproducing an amplified and enriched disc stem cell comprising the stepsof: (a) growing attached nucleus pulposus cells in a first mediumcomprising serum, basic fibroblast growth factor (bFGF), epidermalgrowth factor (EGF), and human neonatal foreskin fibroblast (NFF) cellsupernatant on gelatin-coated tissue culture plates to confluency; (b)isolating a single cell population of nucleus pulposus cells from saidfirst medium; (c) growing said nucleus pulposus cells in suspension in asecond medium comprising methylcellulose, serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) on ultra-low bindingculture plates until at least one of said cells forms a sphere-likecluster, (d) isolating a population of disc stem cells from said secondmedium; (e) growing said disc stem cells in suspension in a third mediumcomprising serum, basic fibroblast growth factor (bFGF), and epidermalgrowth factor (EGF) on ultra-low binding culture plates until at leastsome of said cells form an aggregate cluster, and (f) isolating apopulation of disc stem cells from said third medium, thereby producingan amplified and enriched disc stem cell population.

In another embodiment, the present invention provides an amplified discstem cell population obtained using a method of producing an amplifiedand enriched disc stem cell population comprising the steps of: (a)growing attached nucleus pulposus cells in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates to confluency; (b) isolating apopulation of nucleus pulposus cells from said first medium; (c) growingsaid nucleus pulposus cells in a second medium comprisingmethylcellulose, serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) on ultra-low binding culture plates toconfluency; (d) isolating a population of disc stem cells from saidsecond medium, thereby producing an amplified disc stem cell population.

In another embodiment, the present invention provides an amplified andenriched disc stem cell population obtained using a method of producingan amplified and enriched disc stem cell population comprising the stepsof: (a) suspending isolated nucleus pulposus cells in a mediumcomprising approximately 15% serum, basic fibroblast growth factor(bFGF), epidermal growth factor (EGF), and human neonatal foreskinfibroblast (NFF) cell supernatant; (b) plating the suspension ontogelatin-coated tissue culture plates wherein said cells grow as anattachment culture in a monolayer, thereby producing an amplifiednucleus pulposus cell population, (c) suspending said amplified nucleuspulposus cell population in a medium comprising >10% methylcellulose,fetal calf serum (FCS), basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF); and (d) distributing the suspension ontoultra-low binding culture plates; wherein individual disc stem cellsform grow into spherical shapes in a clonal manner, thereby producing anamplified and enriched disc stem cell population.

In another embodiment, the present invention provides a method oftreating a subject having a herniated disc, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using any of the methods described hereinabove.

In one embodiment, the present invention provides a method of treating asubject having a herniated disc, comprising the step of administering tosaid subject an amplified and enriched disc stem cell populationobtained using a method comprising the steps of: (a) growing nucleuspulposus cells as an attached monolayer in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates wherein said cells grow as anattached monolayer to confluency; (b) isolating a single cell populationof nucleus pulposus cells from said first medium; (c) growing saidnucleus pulposus cells in suspension in a second medium comprisingmethylcellulose, serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) on ultra-low binding culture plates untilthey grow into spherical shapes; (d) isolating a population of disc stemcells from said second medium; (e) growing said disc stem cells insuspension in a third medium comprising serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) on ultra-low bindingculture plates until at least some of said cells form an aggregatecluster, and (f) isolating a population of disc stem cells from saidthird medium, wherein said disc stem cells from said third medium are anamplified and enriched disc stem cell population, thereby producing anamplified and enriched disc stem cell population, thereby treating saidsubject having a herniated disc.

In one embodiment, the present invention provides a method of treating asubject having a herniated disc, comprising the step of administering tosaid subject an amplified and enriched disc stem cell populationobtained using a method comprising the steps of: (a) suspending anucleus pulposus cell population comprising disc stem cells as singlecells at low density in a medium comprising 50% methylcellulose, 5%serum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); (b) distributing the suspension onto ultra-low bindingculture plates; and (c) growing said single cell suspension for 10-20days, wherein an individual disc stem cell grows into a sphere-like cellcluster in a clonal manner, thereby producing an enriched disc stem cellpopulation, thereby treating said subject having a herniated disc.

In one embodiment, the present invention provides a method of treating asubject having a herniated disc, comprising the step of administering tosaid subject an amplified disc stem cell population obtained using amethod of producing an amplified and enriched disc stem cell populationcomprising the steps of: (a) suspending a sphere-like cluster of discstem cells or an isolated disc stem cell from said sphere-like cellcluster in a medium comprising 10% serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF); (b) distributing thesuspension onto ultra-low binding culture plates; (c) growing saidsuspension for 8-16 days, wherein said cluster or cell grows into alarge aggregate cluster of heterogeneous morphology, thereby amplifyingand further enriching an enriched disc stem cell population, therebytreating said subject having a herniated disc.

In one embodiment, the present invention provides a method of treatingdamage to or disease of the spinal joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method comprising the steps of: (a) growingnucleus pulposus cells as an attached monolayer in a first mediumcomprising serum, basic fibroblast growth factor (bFGF), epidermalgrowth factor (EGF), and human neonatal foreskin fibroblast (NFF) cellsupernatant on gelatin-coated tissue culture plates to confluency; (b)isolating a single cell population of nucleus pulposus cells from saidfirst medium; (c) growing said nucleus pulposus cells in suspension in asecond medium comprising methylcellulose, serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) on ultra-low bindingculture plates until at least one of said cells forms a sphere-likecluster, (d) isolating a population of disc stem cells from said secondmedium; (e) growing said disc stem cells in suspension in a third mediumcomprising serum, basic fibroblast growth factor (bFGF), and epidermalgrowth factor (EGF) on ultra-low binding culture plates until at leastsome of said cells form an aggregate cluster; and (f) isolating apopulation of disc stem cells from said third medium, wherein said discstem cells from said third medium are an amplified and enriched discstem cell population, thereby producing an amplified and enriched discstem cell population, thereby treating damage to or disease of thespinal joint of said subject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of the spinal joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method comprising the steps of: (a)suspending a nucleus pulposus cell population comprising disc stem cellsas single cells at low density in a medium comprising 50%methylcellulose, 5% serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF); (b) distributing the suspension ontoultra-low binding culture plates; and (c) growing said single cellsuspension for 10-20 days, wherein an individual disc stem cell growsinto a sphere-like cell cluster in a clonal manner, thereby producing anenriched disc stem cell population, thereby treating damage to ordisease of the spinal joint of said subject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of the spinal joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method of producing an amplified andenriched disc stem cell population comprising the steps of: (a)suspending a sphere-like cluster of disc stem cells or an isolated discstem cell from said sphere-like cell cluster in a medium comprising 10%serum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); (b) distributing the suspension onto ultra-low bindingculture plates; (c) growing said suspension for 8-16 days, wherein saidcluster or cell grows into a large aggregate cluster of heterogeneousmorphology, thereby amplifying and further enriching an enriched discstem cell population, thereby producing an amplified disc stem cellpopulation, thereby treating damage to or disease of the spinal joint ofsaid subject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of a joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) growing nucleuspulposus cells as an attached monolayer in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates to confluency; (b) isolating asingle cell population of nucleus pulposus cells from said first medium;(c) growing said nucleus pulposus cells in suspension in a second mediumcomprising methylcellulose, serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) on ultra-low binding cultureplates until at least one of said cells forms a sphere-like cluster, (d)isolating a population of disc stem cells from said second medium; (e)growing said disc stem cells in suspension in a third medium comprisingserum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF) on ultra-low binding culture plates until at least some ofsaid cells form an aggregate cluster; and (f) isolating a population ofdisc stem cells from said third medium, wherein said disc stem cellsfrom said third medium are an amplified and enriched disc stem cellpopulation, thereby producing an amplified and enriched disc stem cellpopulation, thereby treating damage to or disease of the joint of saidsubject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of a joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) suspending anucleus pulposus cell population comprising disc stem cells as singlecells at low density in a medium comprising 50% methylcellulose, 5%serum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); (b) distributing the suspension onto ultra-low bindingculture plates; and (c) growing said single cell suspension for 10-20days, wherein an individual disc stem cell grows into a sphere-like cellcluster in a clonal manner, thereby producing an enriched disc stem cellpopulation, thereby treating damage to or disease of the joint of saidsubject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of a joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method of producing an amplified and enriched discstein cell population comprising the steps of: (a) suspending asphere-like cluster of disc stem cells or an isolated disc stem cellfrom said sphere-like cell cluster in a medium comprising 10% serum,basic fibroblast growth factor (bFGF), and epidermal growth factor(EGF); (b) distributing the suspension onto ultra-low binding cultureplates; (c) growing said suspension for 8-16 days, wherein said clusteror cell grows into a large aggregate cluster of heterogeneousmorphology, thereby amplifying and further enriching an enriched discstem cell population, thereby treating damage to or disease of the jointof said subject.

In one embodiment, the present invention provides a method of repairingdamaged or diseased disc tissue in a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) growing nucleuspulposus cells as an attached monolayer in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates to confluency; (b) isolating asingle cell population of nucleus pulposus cells from said first medium;(c) growing said nucleus pulposus cells in suspension in a second mediumcomprising methylcellulose, serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) on ultra-low binding cultureplates until at least one of said cells forms a sphere-like cluster, (d)isolating a population of disc stem cells from said second medium; (e)growing said disc stem cells in suspension in a third medium comprisingserum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF) on ultra-low binding culture plates until at least some ofsaid cells form an aggregate cluster; and (f) isolating a population ofdisc stem cells from said third medium, wherein said disc stem cellsfrom said third medium are an amplified and enriched disc stem cellpopulation, thereby producing an amplified and enriched disc stein cellpopulation, thereby repairing damaged or diseased disc tissue in saidsubject.

In one embodiment, the present invention provides a method of repairingdamaged or diseased disc tissue in a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) suspending anucleus pulposus cell population comprising disc stem cells as singlecells at low density in a medium comprising 50% methylcellulose, 5%serum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); (b) distributing the suspension onto ultra-low bindingculture plates; and (c) growing said single cell suspension for 10-20days, wherein an individual disc stem cell grows into a sphere-like cellcluster in a clonal manner, thereby producing an enriched disc stem cellpopulation, thereby repairing damaged or diseased disc tissue in saidsubject.

In one embodiment, the present invention provides a method of repairingdamaged or diseased disc tissue in a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method of producing an amplified and enriched disc stemcell population comprising the steps of: (a) suspending a sphere-likecluster of disc stem cells or an isolated disc stem cell from saidsphere-like cell cluster in a medium comprising 10% serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF); (b)distributing the suspension onto ultra-low binding culture plates; (c)growing said suspension for 8-16 days, wherein said cluster or cellgrows into a large aggregate cluster of heterogeneous morphology,thereby amplifying and further enriching an enriched disc stem cellpopulation, thereby repairing damaged or diseased disc tissue in saidsubject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of a joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) growing nucleuspulposus cells as an attached monolayer in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates to confluency; (b) isolating asingle cell population of nucleus pulposus cells from said first medium;(c) growing said nucleus pulposus cells in suspension in a second mediumcomprising methylcellulose, serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) on ultra-low binding cultureplates until at least one of said cells forms a sphere-like cluster, (d)isolating a population of disc stem cells from said second medium; (e)growing said disc stem cells in suspension in a third medium comprisingserum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF) on ultra-low binding culture plates until at least some ofsaid cells form an aggregate cluster, and (f) isolating a population ofdisc stem cells from said third medium, wherein said disc stem cellsfrom said third medium are an amplified and enriched disc stem cellpopulation, thereby producing an amplified and enriched disc stem cellpopulation, thereby treating damage to or disease of a joint of saidsubject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of a joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) suspending anucleus pulposus cell population comprising disc stem cells as singlecells at low density in a medium comprising 50% methylcellulose, 5%serum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); (b) distributing the suspension onto ultra-low bindingculture plates; and (c) growing said single cell suspension for 10-20days, wherein an individual disc stem cell grows into a sphere-like cellcluster in a clonal manner, thereby producing an enriched disc stem cellpopulation, thereby treating damage to or disease of a joint of saidsubject.

In one embodiment, the present invention provides a method of treatingdamage to or disease of a joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method of producing an amplified and enriched disc stemcell population comprising the steps of: (a) suspending a sphere-likecluster of disc stem cells or an isolated disc stem cell from saidsphere-like cell cluster in a medium comprising 10% serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF); (b)distributing the suspension onto ultra-low binding culture plates; (c)growing said suspension for 8-16 days, wherein said cluster or cellgrows into a large aggregate cluster of heterogeneous morphology,thereby amplifying and further enriching an enriched disc stem cellpopulation, thereby treating damage to or disease of a joint of saidsubject.

In one embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of a spinal joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of: (a) growing nucleuspulposus cells as an attached monolayer in a first medium comprisingserum, basic fibroblast growth factor (bFGF), epidermal growth factor(EGF), and human neonatal foreskin fibroblast (NFF) cell supernatant ongelatin-coated tissue culture plates to confluency; (b) isolating asingle cell population of nucleus pulposus cells from said first medium;(c) growing said nucleus pulposus cells in suspension in a second mediumcomprising methylcellulose, serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) on ultra-low binding cultureplates until at least one of said cells forms a sphere-like cluster; (d)isolating a population of disc stem cells from said second medium; (e)growing said disc stem cells in suspension in a third medium comprisingserum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF) on ultra-low binding culture plates until at least some ofsaid cells form an aggregate cluster; and (f) isolating a population ofdisc stem cells from said third medium, wherein said disc stem cellsfrom said third medium are an amplified and enriched disc stem cellpopulation, thereby producing an amplified and enriched disc stem cellpopulation, thereby preventing, inhibiting, or decreasing the likelihoodof damage to or disease of the spinal joint of said subject.

In one embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of the spinal joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method comprising the steps of (a) suspending a nucleuspulposus cell population comprising disc stem cells as single cells atlow density in a medium comprising 50% methylcellulose, 5% serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF); (b)distributing the suspension onto ultra-low binding culture plates; and(c) growing said single cell suspension for 10-20 days, wherein anindividual disc stem cell grows into a sphere-like cell cluster in aclonal manner, thereby producing an enriched disc stem cell population,thereby preventing, inhibiting, or decreasing the likelihood of damageto or disease of the spinal joint of said subject.

In one embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of the spinal joint of a subject, comprising the step ofadministering to said subject an amplified disc stem cell populationobtained using a method of producing an amplified and enriched disc stemcell population comprising the steps of: (a) suspending a sphere-likecluster of disc stem cells or an isolated disc stem cell from saidsphere-like cell cluster in a medium comprising 10% serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF); (b)distributing the suspension onto ultra-low binding culture plates; (c)growing said suspension for 8-16 days, wherein said cluster or cellgrows into a large aggregate cluster of heterogeneous morphology,thereby amplifying and further enriching an enriched disc stem cellpopulation, thereby preventing, inhibiting, or decreasing the likelihoodof damage to or disease of the spinal joint of said subject.

In one embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of a cartilage-containing joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method comprising the steps of: (a) growingnucleus pulposus cells as an attached monolayer in a first mediumcomprising serum, basic fibroblast growth factor (bFGF), epidermalgrowth factor (EGF), and human neonatal foreskin fibroblast (NFF) cellsupernatant on gelatin-coated tissue culture plates to confluency; (b)isolating a population of nucleus pulposus cells from said first medium;(c) growing said nucleus pulposus cells in a second medium comprisingmethylcellulose, serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) on ultra-low binding culture plates toconfluency; (d) isolating a population of disc stem cells from saidsecond medium; (e) growing said disc stem cells in suspension in a thirdmedium comprising serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) and lacking methylcellulose on ultra-lowbinding culture plates to confluency; and (f) isolating a population ofdisc stem cells from said third medium, wherein said disc stem cellsfrom said third medium are an amplified disc stem cell population,thereby preventing, inhibiting, or decreasing the likelihood of damageto or disease of the cartilage-containing joint of said subject.

In one embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of a cartilage-containing joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method comprising the steps of: (a)suspending a nucleus pulposus cell population comprising disc steincells as single cells at low density in a medium comprising 50%methylcellulose, 5% serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF); (b) distributing the suspension ontoultra-low binding culture plates; and (c) growing said single cellsuspension for 10-20 days, wherein an individual disc stem cell growsinto a sphere-like cell cluster in a clonal manner, thereby producing anenriched disc stem cell population, thereby preventing, inhibiting, ordecreasing the likelihood of damage to or disease of thecartilage-containing joint of said subject.

In one embodiment, the present invention provides a method ofpreventing, inhibiting, or decreasing the likelihood of damage to ordisease of a cartilage-containing joint of a subject, comprising thestep of administering to said subject an amplified disc stem cellpopulation obtained using a method of producing an amplified andenriched disc stem cell population comprising the steps of: (a)suspending a sphere-like cluster of disc stem cells or an isolated discstem cell from said sphere-like cell cluster in a medium comprising 10%serum, basic fibroblast growth factor (bFGF), and epidermal growthfactor (EGF); (b) distributing the suspension onto ultra-low bindingculture plates; (c) growing said suspension for 8-16 days, wherein saidcluster or cell grows into a large aggregate cluster of heterogeneousmorphology, thereby amplifying and further enriching an enriched discstem cell population, thereby preventing, inhibiting, or decreasing thelikelihood of damage to or disease of the cartilage-containing joint ofsaid subject.

In one embodiment, a joint or tissue is diseased, damaged or acombination thereof. Therefore, in one embodiment, a joint isstructurally damaged but not diseased, diseased but not structurallydamaged, or diseased and damaged, which in one embodiment is due to thedestruction of the joint by the disease or because the disease weakensthe joints such that typical forces cause structural damage.

In another embodiment, the present invention provides disc stem cellsand methods of producing disc stem cells that may be used as part of aregenerative or reconstructive therapy for rebuilding the spinal jointafter discectomy. Thus, in one embodiment, the present inventionprovides a method of rebuilding the spinal joint after discectomycomprising: administering to a subject an amplified disc stem cellpopulation obtained using any of the methods described herein.

In another embodiment, the present invention provides a method of stemcell based therapeutics. In another embodiment, the present inventionprovides a method of treating degenerative disc disease.

In another embodiment, the present invention provides a method oftreating a subject having a herniated disc. In another embodiment, thepresent invention provides a method of treating a subject having adegenerative disc disease (DDD). In another embodiment, the presentinvention provides a method of treating a subject having a DDD at onelevel in the lumbar spine (from L3-S1). In another embodiment, thepresent invention provides a method of treating a subject having no morethan Grade 1 spondylolisthesis. In another embodiment, the presentinvention provides a method of treating a subject having more than Grade1 spondylolisthesis. In another embodiment, the present inventionprovides a method of treating a subject having no more than Grade 1spondylolisthesis that have had no relief from pain after at least sixmonths of non-surgical treatment.

In another embodiment, the present invention provides disc cells for useas a therapy that when administered to a subject restores disc height.In another embodiment, the present invention provides disc cells for useas a therapy that when administered to a subject reduces pain. Inanother embodiment, the present invention provides disc cells for use asa therapy that when administered to a subject restores movement at thelevel where it is implanted.

In one embodiment, the present invention provides methods of treatingIDD (intervertebral disc degeneration. In one embodiment, IDD isdiagnosed using a combination of clinical criteria and imaging of thespine. In one embodiment, MRI is used to image the vertebrae and discfor evidence of disc bulging or herniation, endplate degradation(Schmorl's nodes), disc narrowing, tears in the annular disc capsule,osteophytes, compression of the neural elements, and changes in tissuesignal characteristics.

In one embodiment, the present invention provides methods of treating,inhibiting, or suppressing lower back pain, which in one embodiment, ispain in the lumbar-sacral spine. In another embodiment, the presentinvention provides methods of treating, inhibiting, or suppressing neckpain. In another embodiment, the present invention provides methods oftreating, inhibiting, or suppressing cervical spinal pain.

In one embodiment, the present invention provides methods of treating,inhibiting, or suppressing the degeneration of the lumbar-sacral spine.In another embodiment, the present invention provides methods oftreating, inhibiting, or suppressing the degeneration of the cervicalspine.

In one embodiment, cells produced using the methods of the presentinvention may be used along with other compositions to treat thediseases described herein. Thus, in one embodiment, compositions thatenhance the expression of pro-disc genes (or their protein products)such as SOX-9, TGFbeta1, TIMP1, and BMP2 through molecular and genetherapy approaches may be used in conjunction with the cells producedusing the methods of the present invention.

In one embodiment, the present invention is directed to compositions andmethods for the repair and/or replacement of degenerated or damaged ordiseased intervertebral discs through reformation of intervertebral disctissue. By implanting disc stem cells or differentiated nucleus pulposuscells with a carrier into the intervertebral space of a degenerateddisc, the damaged or diseased tissue can effectively be repaired orreplaced.

In one embodiment, “treating” refers to either therapeutic treatment orprophylactic or preventative measures, wherein the object is to preventor lessen the targeted pathologic condition or disorder as describedhereinabove. Thus, in one embodiment, treating may include directlyaffecting or curing, suppressing, inhibiting, preventing, reducing theseverity of, delaying the onset of, reducing symptoms associated withthe disease, disorder or condition, or a combination thereof. Thus, inone embodiment, “treating” refers inter alia to delaying progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. In one embodiment, “preventing”refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, or acombination thereof. In one embodiment, “suppressing” or “inhibiting”,refers inter alia to reducing the severity of symptoms, reducing theseverity of an acute episode, reducing the number of symptoms, reducingthe incidence of disease-related symptoms, reducing the latency ofsymptoms, ameliorating symptoms, reducing secondary symptoms, reducingsecondary infections, prolonging patient survival, or a combinationthereof.

Some embodiments of the invention include methods of treating theinitial stages of degenerative intervertebral disc disease in a subject,and involve minimally invasive surgical techniques, such as theimplantation of a biomaterial scaffold and/or nucleus pulposus cellsinto the nucleus pulposus space of the subject. Biomaterial scaffoldsare described in U.S. Pat. No. 5,964,807, incorporated herein byreference in its entirety.

In one embodiment, disc stem cells or nucleus pulposus cells derivedfrom disc stem cells produced using the methods of the present inventioncan be used in open/classical approaches or by closed/minimally invasiveapproaches to disc treatment. In one embodiment, percutaneous injectionthrough a cannula is used to treat or prevent in situ degenerative discdisease without structural damage. In one embodiment, percutaneousinjection is an outpatient treatment.

In one embodiment, disc stem cells are delivered to a subject in need.In another embodiment, disc stem cells and a cell carrier, such as, inone embodiment, Hystem-HP, from Glycosan, Inc., made of synthetic HA, isdelivered to a subject in need. In another embodiment, aggrecan,amniotic matrix, etc is delivered with disc stem cells to a subject inneed. In another embodiment, growth factors (in one embodiment, FGF-2,TGFB3) are delivered with disc stem cells to a subject in need. Inanother embodiment, additional supplements are delivered with disc steincells to a subject in need.

Some embodiments of the invention involve implanting a biomaterialscaffold directly into the nucleus pulposus space with one or morepercutaneous injections. In some embodiments of the invention, thebiomaterial scaffold comprises biologically active glass, as previouslydescribed. In some embodiments of the invention, the scaffold furthercomprises biologically active molecules. In some embodiments of theinvention, the scaffold is combined with one or more pharmaceuticallyacceptable excipients prior to implantation into the nucleus pulposusspace. Pharmaceutically acceptable excipients are familiar to theskilled artisan and include, but are not limited to, buffers,physiological saline, and viscous fluids that harden into a gelatinouscomposite, such as, for example, self-setting hydrogel and alginate.Implantation of the biomaterial scaffold into the nucleus pulposus spaceleads to regeneration of nucleus pulposus cells with concomitantrestoration of the function of the nucleus pulposus tissue.

In one embodiment of the invention, the methods of the present inventionfurther comprise preparing cells, mixing cells with extracellularmatrix, growth factors, or a combination thereof, loading or attachingcells to a cell carrier, injecting the cells and cell carrier into thedisc, placing cells into a disc during surgery after the discectomy, ora combination thereof.

Some embodiments of the invention involve implanting nucleus pulposuscells into the nucleus pulposus space of a degenerated disc of a subjectby making one or more injections with a needle, which in one embodiment,is a percutaneous injection. Ultrasound or other imaging techniques canbe used to guide the needle to the nucleus pulposus space. In someembodiments of the invention, after implantation into the nucleuspulposus space, the nucleus pulposus cells continue to proliferate andexpand, thereby regenerating nucleus pulposus tissue and reestablishingthe natural function of the degenerated disc.

In another embodiment, administration of disc stem cells, with orwithout a carrier is via direct access to the joint, which in oneembodiment, is part of a surgical procedure. In one embodiment, thesurgical procedure is a conventional open discectomy or microdiscectomy.In one embodiment, the surgical procedure is performed on a subject whohad spinal joint disease, which in one embodiment, is structural withinvolvement of a nerve root and, in one embodiment involves severe painin an extremity and, optionally back pain. In one embodiment, thesurgical procedure would be performed after failure of conservativecare. In one embodiment, the surgeon has to damage the joint to getaccess to the herniated disc (the structural defect). In one embodiment,a portion of the disc is removed leaving a defect in the disc andannulus (discectomy). In one embodiment, the surgeon applies the discstem cell therapeutic product directly to the disc defect immediatelyafter the discectomy, and then closes the wound.

In some embodiments of the invention, the nucleus pulposus cells arecombined with one or more pharmaceutically acceptable excipients, asdescribed above, prior to implantation into the nucleus pulposus space.In some embodiments of the invention, the nucleus pulposus cells arecombined with biologically active molecules prior to implantation intothe nucleus pulposus space.

In some embodiments of the invention, nucleus pulposus cells aregenerated by culturing nucleus pulposus cells and/or precursor cells,and the cells are then implanted into the nucleus pulposus space of adegenerated disc of a subject to be treated. In some embodiments of theinvention, following cell culture, and prior to implantation into thenucleus pulposus space, contaminating non-nucleus pulposus cells areremoved from the exogenously-cultured nucleus pulposus cells usingmethods familiar to one of ordinary skill in the art. In someembodiments of the invention, the exogenously cultured nucleus pulposuscells are removed from the carrier material upon which they were seededduring culture prior to implantation of the cells into the nucleuspulposus space.

Some embodiments of the invention involve methods of treating theadvanced stages of intervertebral disc disease in a subject. Someembodiments of the invention involve implanting nucleus pulposus cellsinto the nucleus pulposus space as part of a larger substrate, whichincludes, in some embodiments of the invention, carrier material uponwhich the cells were seeded during culture.

In accordance with some embodiments of the present invention, thecarrier is biodegradable, which means that, after implantation ofnucleus pulposus cells into a degenerated disc, the carrier degradesinto natural, biocompatible byproducts over time until the carrier issubstantially eliminated from the implantation site and, ultimately, thebody. In accordance with some embodiments of the present invention, therate of biodegradation of the carrier is less than or equal to the rateof intervertebral disc tissue formation such that the rate of tissueformation is sufficient to replace the carrier that has biodegraded.

In some aspects of the present invention, the biodegradable carrier isbioactive, which means that the carrier enhances cell function. Forinstance, bioactive glass granules have been shown to enhance cellgrowth of typical bone cells. Schepers et al., U.S. Pat. No. 5,204,106.In addition, dense bioactive glass discs have been found to enhanceostoprogenitor cell differentiation beyond the levels of enhanceddifferentiation elicited by bone morphogenic protein. H. Baldick, etal., Transactions 5th World Biomaterials Conference, Toronto, II-114(June, 1996).

In some embodiments of the invention, the biodegradable carrier hassufficient mechanical strength to act as a load bearing spacer untilintervertebral disc tissue is reformed. In some embodiments, thebiodegradable carrier is biocompatible such that it does not elicit animmune or inflammatory response that might result in rejection of theimplanted material.

In some embodiments of the invention, the nucleus pulposus space of thedegenerated disc to be treated by the methods of the invention isevacuated prior to implantation of the nucleus pulposus stem cells.Preferably, for treatment of advanced stages of intervertebral discdisease, the nucleus pulposus space of the degenerated disc is evacuatedprior to implantation of the nucleus pulposus cells.

Evacuation of the degenerated intervertebral disc tissue, and primarilythe nucleus pulposus tissue, is performed using known surgical toolswith procedures adapted to meet the needs of the present invention. Forexample, an incision or bore may be made at the lateral edge in theannulus fibrosus and the intervertebral disc tissue is extracted fromthe nucleus pulposus via, for example, the guillotine cutting approach.The tissue can be extracted using a pituitary puller, a kerrisonrongeur, scalpel, bore, or curette. Alternatively, the tissue may beaspirated. In some embodiments, the annulus fibrosus, or significantportions thereof, is left intact. It is preferred in some embodiments ofthe invention that at least 50% of the annulus fibrosus remains intact.It is more preferred in some embodiments that at least 85% of theannulus fibrosus remains intact.

Where delay occurs between evacuation of nucleus pulposus tissue andimplantation of the exogenously cultured nucleus pulposus cells, theevacuated space may be temporarily filled with gel foam or other loadbearing spacers known in the art.

In some embodiments of the invention, the previously described methodsfor treating intervertebral disc disease are used in conjunction withother known, conventional treatments.

The materials, methods and examples presented herein are intended to beillustrative, and are not intended to limit the scope of the invention.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Unlessotherwise defined, all technical and scientific terms are intended tohave their art-recognized meanings.

In another embodiment, the present invention provides a kit foramplifying a disc stem cell population comprising a container comprisinga cluster medium comprising serum, basic fibroblast growth factor(bFGF), and epidermal growth factor (EGF) and lacking methylcelluloseand instructions for the use thereof.

In another embodiment, the present invention provides a kit forproducing an enriched disc stem cell population from nucleus pulposuscells comprising a container comprising a discosphere medium comprisingmethylcellulose, serum, basic fibroblast growth factor (bFGF), andepidermal growth factor (EGF) and instructions for the use thereof.

In another embodiment, the present invention provides a kit foramplifying a population of nucleus pulposus cells comprising a containercomprising a Discotek medium comprising serum, basic fibroblast growthfactor (bFGF), epidermal growth factor (EGF), and human neonatalforeskin fibroblast (NFF) cell supernatant and instructions for the usethereof.

In another embodiment, the present invention provides a kit forpreparing disc tissue comprising a medium for disc preparationcomprising PBS supplemented with antibiotics and/or antimycotics,DMEM/F12 medium comprising Collagenase II, Trypsin, and instructions forthe use thereof.

It is to be understood that a kit of the present invention may compriseany one or more of the kits described hereinabove and is to beconsidered a part of the invention.

It is to be understood that a kit of the present invention may comprisea single medium or various combinations thereof. Therefore a kit of thepresent invention may comprise, for example, a first containercomprising a cluster medium and, optionally, a second containercomprising a discosphere medium, and, optionally, a third containercomprising Discotek medium and, optionally, a fourth containercomprising a disc preparation medium. It is to be further understoodthat each component of a described medium may be provided within aseparate container to be mixed in the medium according to theinstructions provided at a specified step in the protocol described inthe instructions, in one embodiment. In one embodiment, the media isDMEM/F12 and, optionally N10 or N2 media.

In one embodiment, “instructional material,” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions of the inventionin the kit.

In one embodiment, the instructional material of the kit may, forexample, be affixed to a container that contains the composition (e.g.,media, media components, and the like) of the invention or be shippedtogether with a container which contains the composition. Alternatively,the instructional material may be shipped separately from the containerwith the intention that the recipient uses the instructional materialand the composition cooperatively.

In another embodiment, the kit further comprises wash solutions,reagents for marker detection, which in one embodiment, is markers ofdisc stem cells, which are known in the art.

Other kits, useful for practicing the invention as disclosed herein, areencompassed herein. The above discussed kits are exemplary and otherkits as would be readily appreciated by the skilled artisan, based uponthe disclosure provided herein, are included in the invention.

In another embodiment, the present invention provides a kit forcultivating disc stem cells, maintaining the pluripotency of disc stemcells, amplifying disc stem cells, or a combination thereof.

In one embodiment, the present invention provides a disc stem cellculture platform that enables isolation, culture, and expansion of discstem cells from adult disc tissue, as described herein.

In another embodiment, the present invention provides a medium forcultivating and amplifying disc stem cells comprising: DMEM/F12, NFF,serum, FGF, and EGF. In another embodiment, the present inventionprovides a medium for cultivating and amplifying disc stem cellscomprising: 66% DF10, 33% NFF, 14.5% serum, 10 ng/ml FGF, and 10 ng/mlEGF. In one embodiment, this media is referred to herein as Discotekmedia.

In another embodiment, the present invention provides a disc replacementdevice comprising nucleus pulposus cells. In another embodiment, thepresent invention provides an artificial disc comprising nucleuspulposus cells. In another embodiment, the disc replacement device is anintervertebral disc replacement device. Methods for making and usingthese compositions are known in the art and described in for e.g. US2009/0074835, which is incorporated by reference herein in its entirety.

In another embodiment, an intervertebral disc is located between theconcave articular surfaces of the adjacent vertebral body endplates. Inanother embodiment, the disc replacement device of the present inventionpermits movements such as flexion, extension, lateral flexion, androtation. In another embodiment, the disc replacement device of thepresent invention is used to repair and/or replace injured or damaged ordiseased intervertebral discs. In another embodiment, the discreplacement device of the present invention provides a prosthetic discthat combines both stability to support the high loads, of the patient'svertebrae and flexibility to provide the patient with sufficientmobility and proper spinal column load distribution.

In another embodiment, the present invention provides a method ofproducing an artificial disc, comprising the step of growingdiscospheres or clusters in a disc scaffold. In another embodiment, thepresent invention provides a method of producing an intervertebral discreplacement device, comprising the step of growing discospheres orclusters in a disc scaffold. In another embodiment, discospheres orclusters are administered onto a disc scaffold. In another embodiment,discospheres or clusters are administered into a layer comprisingcollagen in the disc scaffold. In another embodiment, discospheres orclusters are administered onto a layer comprising collagen in the discscaffold. In another embodiment, discospheres or clusters are injectedinto a disc scaffold (Example 4). In another embodiment, discospheres orclusters are injected onto a disc scaffold. In another embodiment,discospheres or clusters are injected into a layer comprising collagenin the disc scaffold. In another embodiment, discospheres or clustersare injected onto a layer comprising collagen in the disc scaffold. Inanother embodiment, the discospheres or clusters of the presentinvention are applied or injected into or onto the disc scaffoldtogether with a composition of the present invention. In anotherembodiment, the discospheres or clusters of the present invention areapplied or injected into or onto the disc scaffold together with aDMEM/F12 medium supplemented with 10% FCS.

In another embodiment, the present invention provides a method ofbiologically repairing a disc, comprising the step of growingdiscospheres or clusters in culture, and resuspending them at the timeof surgery in a cell carrier.

In another embodiment, a spinal disc tissue of the present inventioncomprises matured nucleus pulposus cells derived from disc stem cells ofthe present invention attached to a disc scaffold of the presentinvention. In another embodiment, a spinal disc tissue of the presentinvention comprises fibroblasts and matured nucleus pulposus cellsderived from discospheres of the present invention attached to a discscaffold of the present invention.

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprising coatingthe disc scaffold of the present invention with nucleus pulposus cellsgrowth factors. In another embodiment, the present invention provides amethod of producing an intervertebral disc replacement device,comprising coating the disc scaffold of the present invention withnucleus pulposus cells adhesion factors. In another embodiment, thepresent invention provides a method of producing an intervertebral discreplacement device, comprising coating the disc scaffold of the presentinvention with nucleus pulposus cells differentiation factors. Inanother embodiment, the present invention provides a method of producingan intervertebral disc replacement device, comprising placing the discscaffold of the present invention in a medium comprising nucleuspulposus cells growth factors, adhesion factors, and differentiationfactors.

In one embodiment, the nucleus pulposus cells provided by the presentinvention comprise autograft nucleus pulposus cells, allograft nucleuspulposus cells, or xenograft nucleus pulposus cells.

In another embodiment, the present invention provides that the recipientof the nucleus pulposus cells of the present invention is the donor. Inanother embodiment, the present invention provides that the recipient ofthe nucleus pulposus cells of the present invention may function atleast in part as a donor. In another embodiment, the present inventionprovides that the donor of nucleus pulposus cells of the presentinvention is a single donor. In another embodiment, the presentinvention provides that multiple donors provide nucleus pulposus cellsof the present invention to a single recipient. In another embodiment,the present invention provides that multiple donors provide nucleuspulposus cells of the present invention to multiple recipients. Inanother embodiment, the present invention provides that fetal sourcesare used. In another embodiment, the present invention provides that thedonor or donors of the nucleus pulposus cells of the present inventionis or are preferably having a familial relationship to the recipient inorder to minimize or avoid immunosuppression. In another embodiment, thepresent invention provides that the donor or donors of the nucleuspulposus cells of the present invention is or are preferably having afamilial relationship to the recipient in order to minimize or avoid theneed for immunosuppressive substances. In another embodiment, thepresent invention provides guidelines for tissue procurement includingsurgical techniques of removal, number of hours between death of thedonor and tissue procurement, and testing of the donor for infectiousdisease, are well known to one of skill in the art.

In one embodiment, two decades of direct and indirect evidence inanimals and humans provide support that allograft tissue will not beimmunorejected in recipients and immunotyping will not be required.

In one embodiment, cell culture behavior is relative to the tissuesource, which in one embodiment, refers to the species of the tissuesource, the age of the tissue source, the pathobiology of the tissue, ora combination thereof. In one embodiment, the time to confluency fordisc tissue from healthy, young subjects and/or from tissue that is morecellular is 3-5 days. In one embodiment, the time to confluency for disctissue from older animals, from certain species such as bovine, adultporcine, aged human or from degenerated tissue may be longer than 3-5days.

In one embodiment, a robust culture in a T75 Flask yields around 10×10⁶cells.

In one embodiment, cells are not passaged back into Attachment culture,because, in one embodiment, each subsequent passage results in adecreased percentage of cells in the stem cell fraction due todifferentiation of stem cells during the Attachment culture.

In one embodiment, the phrases fetal calf serum and fetal bovine serummay be used in the methods interchangeably.

In one embodiment, the methods of the present invention provide methodsof procuring disc tissue, methods of processing disc tissue, methods ofculturing disc cells, and methods of culturing disc stem cells, as weredescribed herein. In another embodiment, the methods of the presentinvention provide methods of preparing disc stem cells for clinical use,which in one embodiment, comprise combining disc stem cells with growthfactors and matrix molecules and re-suspending them in a cell carrier.In another embodiment, the methods of the present invention providemethods of treating, suppressing or inhibiting disc disease or symptomsthereof comprising administering disc stem cells or nucleus pulposuscells prepared using the methods described herein. In one embodiment,administering comprises injected a stem cell composition of the presentinvention percutaneously under CT or Fluoro guidance into the disc spacein the context of nonstructural degenerative disc disease, whether one,two, or several levels. In another embodiment, in the context ofstructural disc disease requiring a surgery, the stem cell compositionis injected through the annular defect into the disc defect left by thesurgical discectomy.

EXPERIMENTAL DETAILS SECTION Materials and Methods

Preparation of Disc Tissues

A biopsy specimen of human nucleus pulposus was cut in piecesapproximately 2-3 mm in size. The pieces were collected into a 50 mlfalcon tube containing 30 ml of Phosphate buffered saline (PBS)supplemented with antibiotics/antimycotics. The tube was thencentrifuged to pellet the tissue pieces, and PBS was aspirated. Thetissue pellet was weighed per protocol. 15 ml of Dulbecco's ModifiedEagle Media with F12 (DMEM/F12) medium containing 300 units/ml ofCollagenase II solution was added to the tube. The contents of the tubewere transferred to a T75 tissue culture flask, which was placedhorizontally in a tissue culture incubator to incubate for 24 h. After24 hours of enzymatic digestion, the contents of the T75 Flask weretransferred to a 50 ml falcon tube. At that time, the tissue wascompletely digested, although floating cellular aggregates resistant todigestion were occasionally found.

The 50 ml falcon tube was centrifuged at 1200 rpm to pellet the cells,and the supernatant aspirated. The pellet was suspended in 2 ml of warmTrypsin. The suspension was incubated at 37° C. for two minutes. Thesuspension was triturated with a fire polished Pasteur pipette to ensurea true single cell suspension. 0.5 ml of Fetal Bovine Serum was added tostop the action of Trypsin.

A cell count was then performed. Next, the suspension was centrifuged at1200 rpm to pellet the cells. The supernatant was aspirated and the cellpellet was resuspended in culture media to achieve the desired cellconcentration.

Attachment Culture

Human Neonatal Foreskin Fibroblast Conditioned Media (Discotek Media)

Materials

Preparing human neonatal foreskin fibroblasts (NFF). Foreskin specimenswere cut into approximately 2-3 mm pieces, and the fragments weretransferred to a 50 ml plastic tube in 30 ml of PBS supplemented withantibiotic-antimycotic. The tissue pieces were centrifuged at 1000 rpmfor 5 min. PBS was aspirated, then 1-3 ml of Collagenase II solution(300 U/ml) in DMEM/F12 medium was added. Tubes were placed in ahorizontal position into a shaker incubator (T=37° C.). Tubes wereshaken at 100 rpm for 3-24 hours until fragments of tissue were visiblydissociated. Trypsin was added post-incubation, and the cells wereincubated for 5 min with or without trituration as needed to completelydissociate the tissue. The resulting cell suspension was filteredthrough a 0.22 micron nylon mesh into a 50 ml tube,

resulting in a single-cell suspension. The cell suspension wascentrifuged for 4 min at 400 g at room temperature and the supernatantremoved by aspiration. Cells were resuspended in DMEM/F12 medium with10% fetal bovine serum (FBS).

NFF Supernatant

Human neonatal foreskin fibroblast (NFF) cells were expanded in DF10media to fill one T150 culture flask to 90% confluency. Next, thesupernatant was collected every two days over 6 days, and filteredthrough a 0.22 um mesh filter. Supernatant was frozen at −20° C. in 50ml aliquots. Media can be stored at −20° C. for up to 3 years.

Methods

Attachment Culture. 0.1% Gelatin coated tissue culture plates wereprepared. The single cell suspension from the disc tissue preparationprotocol was centrifuged at room temperature for 5 min at 1200 rpm. Thesupernatant was then removed by aspiration. The cells were resuspendedin Discotek media (74 ml DF10 media; 6 ml FBS; 40 ml human neonatalforeskin fibroblast (NFF) supernatant (see materials hereinabove); 600μl Penicillin/Streptomycin; 12 μl of 100 ng/uL epidermal growth factors(EGF), final [c]=10 ng/mL); 12 μl of 100 ng/uL fibroblast growthfactor-2 (FGF-2), final [c]=10 ng/mL); total: 120.64 ml) at a volume of10 ml, at a cell surface density of 50,000 cells/cm². The cellsuspension was plated onto the Gelatin coated tissue culture plates.Media and growth factors were changed every second day.

The cells were passaged when they were 90% confluent by washing theattachment culture twice with PBS, and adding Trypsin. The culture withTrypsin was incubated for 5 minutes at 37° C. DMEM supplemented withserum was added to stop the Trypsin digestion. The contents of theplates were transferred to a 50 ml falcon tube. The contents weretriturated to ensure a single cell suspension. A cell count wasperformed.

Cryopreservation

A portion of the cells were then cryopreserved in aliquots of 1 ml/tubein freezing media (90%, 10% DMSO), at 1-3×10⁶ cells/ml.

Discosphere Stem Cell Culture Method-1 (Starting with CryopreservedAttachment Cells)

20 ml of 2% methylcellulose (MC) and 2 mL of FBS were added to two 50 mlFalcon tubes. 10 ml of DF10 medium were added to a 15 ml Falcon tube. 3M frozen cells/ml were thawed, placed in the tube with media, and mixedby inversion. The cell suspension was centrifuged at 1200 rpm for 5minutes at 4° C. The supernatant was aspirated. The cell pellet wasresuspended in 40 ml of 2× N5 medium, and mixed well. 20 mL of thesuspension was added to each of the Falcon tubes. FGF2 and EGF wereadded to each to a final concentration 10 ng/ml each. Tubes were closedtightly, and the contents were carefully mixed by inversion. The mixturewas distributed to 10 cm ultra-low binding culture dishes, 12 ml/dish,and at a concentration of 1×10⁵/ml. Plates were incubated in the tissueculture incubator for continued culture. EGF/FGF was added at 10 ng/mlevery second day. After 12-20 days, well developed discospheres werepresent, and the culture was ready for passage into suspension cultureor for cycling. Cells can be passaged as single cell suspension, or ascollected spheres and transferred to the cluster culture.

Discosphere Stem Cell Culture Method-2 (Starting with AttachmentCulture)

Supernatant was removed and the plate washed with 2× with PBS. 1 mlTrypsin was added. The plate was placed in an incubator for 5 min at 37°C. Plates were removed and an equal amount of DF10 (to inactivateTrypsin) was added. All cell preparations were transferred to a 50 mlFalcon tube. A Pasteur pipette was used to triturate the cells and breakup clusters. A cell count was done. Tubes were centrifuged for 5 min at1200 rpm at room temperature. The supernatant was removed by aspiration.Cells were resuspended in methylcellulose (mix by vortexing) plus 12%fetal calf serum (FCS), diluted with 2× N2 medium to a finalconcentration of 6% serum and a final density of 1×10⁵ cells/ml. EGF andFGF2 were added at 10 ng/ml each, and gently mixed. Solution wasdistributed into ultra-low binding 6-well plates (2 ml/well) coveredwith anti-adhesive coating, and incubated at 37° C. After two weeks,well developed discospheres were present, and the culture was ready forpassage into suspension culture, or for cycling. Cells can be passagedas single cell suspension, or as collected spheres and transferred tothe cluster culture.

Cluster Cell Culture Method

Contents were collected from 6 well plates and transferred to a 50 mlFalcon tube (1 tube/plate). 10 ml of DMEM media was added to the plate,which was washed carefully to remove remaining spheres. Washes wereadded to the falcon tubes. This step was repeated a second time. Tubeswere centrifuged at 1200 rpm for 5 min at 4° C. to pellet the spheres.The supernatant was aspirated, resuspended in 1 ml of Trypsin, andincubated at 37° C. for 5 min. A Pasteur pipette was used to triturateand break up cell clusters. A cell count was performed. Samples werethen centrifuged for 5 min at 1200 rpm at RT, and the supernatant wasaspirated. The cells were re-suspended in N10 media at a density of1×10⁴ cells/ml. Disc tissue cell suspension was then plated on ultra lowbinding plates. EGF and FGF2 were added to a final concentration 10ηg/mleach, and mixed gently. Media was changed every third day. Maturecluster cells were passaged when they were around 150-300 microns indiameter. Cycling back into this culture system was continued up tothree times or until the desired cell number was achieved.

EXAMPLE 1 Discotek Culture Platform

In one embodiment, the Discotek Culture Platform comprises the steps of(1) preparing a single cell suspension from a disc tissue specimen (FIG.1, reference numerals 1-3), (2) the Attachment Culture Method (FIG. 1,reference numerals 4-7), (3) the Discosphere Culture Method (referencenumerals 8-13), and (4) the Cluster Culture Method (FIG. 1, referencenumerals 14-17).

Disc tissue was procured from a human patient during surgery, or from anexperimental animal model (FIGS. 1-2, reference number 1). The tissuewas processed by dissection and then enzymatically digested overnightfor 24 hours at 37° C. in Collagenase II (FIGS. 1-2, reference number2). The result was a single cell suspension of all cells that comprisethe disc tissue that can be plated into the subsequent culture steps(FIGS. 1-2, reference number 3).

The single cell suspension was plated into the attachment culture(reference number 4), in which the plates were coated with gelatin(reference number 5), causing the single cell suspension to attach(reference number 6) as it settles to the culture flask floor andcontacts the surface (reference number 7). The cultures then growefficiently as a monolayer, reaching confluency in 4-7 days when thestarting disc tissue material was derived from young healthy disc tissuespecimens. All species and even degenerated tissues grow well with thisculture method, but the rate of growth varies and is delayed withtissues from older patients or animals, and degenerated tissues, andcertain species (i.e., rat, rabbit, neonatal porcine, young healthyhuman tissue), as compared to specimens derived from humans that areolder, degenerated tissues from any species, or certain other speciesthat are more acellular generally speaking (i.e, adult porcine, cow,etc.)

When the cells were confluent, the attachment culture was passaged bypreparing a single cell suspension using enzymatic digestion, andplating the cells in the Discosphere Culture Method (reference numerals8-11). This was also a step from which to cryopreserve cells for futureresearch studies. The Discosphere culture method has methylcellulose aspart of its media, and uses tissue culture plates that were coated sothat the surfaces were ultra-low binding. Thus, this was a suspensionculture. The cells were plated in low density and because they were insuspension and thus could not attach to each other to the culturevessel, each stem cell grows into a sphere-like cell cluster, in aclonal manner (reference numerals 12-13). Conversely, mature cells andlate progenitors cannot grow without contact of other cells or a culturesurface, and thus they die via apoptosis. The time it takes for thespheres to become mature in size (about 150-300 microns in diameter) wastypically 10-20 days. Tissues that are degenerated, certain species thatare acellular, and tissue from older patients or animal models tooklonger to reach maturity.

When the disc stem cell spheres (Discospheres) from the previouslydescribed culture method were mature, they were then passaged in one oftwo manners. First, to expand the cell population, they were passagedinto the Cluster Culture Method by preparing a single cell suspensionusing enzymatic digestion, and plating the cells in the Cluster CultureMethod at a predefined plating density of 1×10⁴. This was an appropriatetime to cryopreserve stem cells in a single cell form. The cells werethen grown in the Cluster Culture Method in cycles to expand them asneeded. The time it typically takes for passaged cells from a singlecell suspension to reach maturity as cluster-like cell aggregates, wasaround 8-16 days. The end product of cycling the Cluster cells was astem cell product of the culture method, which can be cryopreserved foruse at a future date as indicated. The second passaging approach was totransfer the Discospheres to the Cluster Culture directly. The spheresgrow into large clusters of heterogeneous morphology. The time for thelarge clusters to reach maturity was 8-14 days. This was a stem cellproduct of the culture method, and the large cell clusters can becryopreserved for future use. Tissues that are degenerated, certainspecies that are acellular, and tissue from older patients or animalmodels took longer to reach maturity.

EXAMPLE 2 Preparation of Single Cell Suspensions

Human disc tissues were dissected into smaller pieces, and washedthoroughly of blood and other tissue contaminants (FIG. 3A). Thedissected fragments were placed in media with Collagenase II at 300units/ml and incubated for 24 hours at 37° C. (FIG. 3B). A large amountof debris was present that made counting cells difficult and was removedwith the washing step. A cell strainer was not used because of theroutine presence of cellular clusters that are potentially notochordalclusters that do not break up without further trypsinization andtrituration. It was thought that these cell clusters were a criticalsource of notochordal cells and that they resist digestion due to theirextensive extracellular matrix. A single cell suspension was the productof this method (FIGS. 3C-D). This method does not use Trypsin forprolonged digestion, and does not use mechanical disassociation oftissues, to protect the fragile disc stem cell population. Thisincreases the cellular health of individual stem cells, and results inoverall higher yields of stem cells from the tissues. The disc tissuefrom adult humans and porcine species is very acellular, and theproportion of disc stem cells in a given unit of tissue was quite low.Further, the amount of cells and stem cells in degenerated tissues waseven lower. Because a critical cell density (˜1000-10,000 disc cells/ml)is required to provide enough stem cells to push a stem cell cultureinto active growth and expansion, every possible attempt to stabilize,protect, and enhance the stem cell population was taken. The fraction ofcells that stem cells represent against the total cell population is notknown, but indirect evidence for disc tissue stem cells, and previousadult tissue stem cell models collectively would suggest 0.1-0.001 or1/1000 to 1/100,000.

Porcine disc tissues (FIG. 4A) were dissected into smaller pieces, andwashed thoroughly of blood and other tissue contaminants (FIG. 4B). Thedissected fragments were placed in media with Collagenase at 300units/ml and incubated for 24 hours at 37° C. A single cell suspensionand was the product of this method (FIG. 4C). A cell strainer was notused because of the routine presence of cellular clusters that do notbreak up without brief trypsinization and trituration. It was thoughtthat these cell clusters were a critical source of notochordal cells andthey resist digestion due to their extensive extracellular matrix.

EXAMPLE 3 Attachment Culture Method

The single cell suspension was plated into the Attachment Culture Method(FIG. 5), in which the plates were coated with gelatin, causing thesingle cell suspension to attach as it settles to the culture flaskfloor and contacts the surface (FIGS. 1 and 5, reference numerals 4-5).The cultures then grew efficiently as a monolayer reaching confluency in4-7 days when the starting disc tissue material was derived from younghealthy disc tissue specimens. All species and even degenerated tissuesgrow well with this culture method, but the rate of growth varies and isdelayed with tissues from older patients or animals, and degeneratedtissues, and certain species (i.e., rat, rabbit, neonatal porcine, younghealthy human tissue), as compared to specimens derived from humans thatare older, degenerated tissues from any species, or certain otherspecies that are more acellular generally speaking (i.e., adult porcine,cow, etc.) When the cells were confluent, the attachment culture waspassaged by preparing a single cell suspension using enzymaticdigestion, and plating the cells using the Discosphere Culture Method(FIGS. 1 and 5, reference numerals 6-7). This was also a step from whichto cryopreserve cells for future research studies.

TABLE 1 Key Components of the Attachment Culture Method and DiscotekMedia. COMPONENT AMOUNT/OTHER MEDIA DMEM/HAMSF12 NFF   33% SERUM 14.5%FGF 10 ng/ml EGF 10 ng/ml CELL SURFACE GELATIN

TABLE 2 Identification of important cell number and growth kineticparameters from the tissue preparation method and the Attachment CultureMethod. COMPONENT Number/Other Cell State ATTACHED Time to Confluency(Healthy Cellular Tissues) 5-14 days Time to Confluency (DegeneratedAcellular Tissues) 8-21 days Plating Density 50 × 10³/cm²

Porcine disc tissue cells plated on gelatin coated surfaces in Discotekmedia (Table 1) from the single cell suspension product of the tissuepreparation method show initial attachment (FIG. 6), reached 70%confluency after a 3-5 day incubation (FIG. 7), and confluency after a5-7 day incubation (FIG. 8). Human disc cells reached confluency after10-12 days (FIG. 9). Thus, the rate of growth of the disc cells in thisculture method (time to confluency) was typically dependent on thespecies of the disc tissue, the age of the animal, and the nature of thetissue itself (i.e., healthy vs. the degree to which the disc tissue wasdegenerated). The cell number and growth kinetic parameters from thetissue preparation method and the Attachment Culture Method are shown inTable 2.

Notochordal cells were identified after plating of the single cellsuspension product of tissue preparation using the Attachment CultureMethod before and after cell attachment (FIG. 10). Notochordal cellswere distinctly recognized because of their atypically large size andvacuolated appearance. All three photomicrographs in FIG. 10 show thepredominant cells to be nucleus pulposus cells, but this datademonstrates the presence and persistence of the notochordal cellsinitially and out to several days in this initial culture method.Additionally, FIGS. 10A, 10B, and 10C demonstrate the presence ofseveral types of cells that were not readily definable.

EXAMPLE 4 Discosphere Culture Method

When the cells were confluent, the attachment culture was passaged bypreparing a single cell suspension using enzymatic digestion, andplating the cells in the Discosphere Culture Method (FIG. 11, referencenumerals 1-5). This was also a step from which to cryopreserveattachment cells for future research studies. The Discosphere CultureMethod uses methylcellulose in its media (Table 3), and uses coatedtissue culture plates with ultra-low binding surfaces to create asuspension culture. The cells were plated at low density and becausethey were in suspension and could not attach to the culture vessel, eachcell grew into a sphere-like cell cluster in a clonal manner. Thespheres matured in size typically after 10-20 days. The cell number andgrowth kinetic parameters from the Discosphere Culture Method arepresented in Table 4. When the spheres from the Discosphere CultureMethod were mature, they were then passaged typically by preparing asingle cell suspension (FIG. 11, reference numerals 6-7). However, asecond method that was used was to transfer the disc stem cell spheresdirectly to the cluster culture.

TABLE 3 Key Components of the Discosphere Culture Method and N5 Media.COMPONENT AMOUNT/OTHER MEDIA DMEM/F12 METHYLCELLULOSE 50% SERUM  5% FGF10 ng/ml EGF 10 ng/ml CELL STATE SUSPENSION PLATING CELL DENSITY 1 × 10⁴TISSUE CULTURE SURFACE ULTRA LOW BINDING

Protocol for 100 ml of N5 Concentrations of Reagents: 50 mlMethylcellulose 16.1 μg/ml of Putrescine 45 ml of stock 2X DMEM F12Media (100 μM) 5 ml of Fetal Calf Serum 62.8 μg/ml of Progesterone 100μl of stock 1000X Sodium Selenite (20 μM) 100 μl of stock 1000X Insulin5.2 μg/ml of Sodium Selenite 100 μl of stock 1000X Putrescine (30 μM)100 μl of stock 1000X Progesterone 50 μg/ml of Transferrin. 100 μl ofstock 1000X Transferrin 5 μg/ml of Insulin

TABLE 4 Identification of important cell number and growth kineticparameters from the Discosphere Culture Method. COMPONENT Number/OtherCell State Suspension as Spheres Time to Maturity (Healthy CellularTissues) 10-16 days Time to Maturity (Degenerated Acellular Tissues)10-28 days

When the cells were confluent, the attachment culture was passaged bypreparing a single cell suspension using enzymatic digestion, andplating the cells in the Discosphere Culture Method (FIG. 12). Thespheres that developed were created from a single stem cell clonally. Asthe spheres grew in culture, the outer layers matured somewhat. However,as late progenitors or terminally differentiated stages of developmentwere encountered, the cells entered apoptosis due to the inhospitableculture environment for mature or differentiated cells. Reasons for celldeath were attributed to the lack of attachment to a plate surface, andthe lack of cell-cell interaction caused by a relative low platingdensity.

Discospheres were collected from the culture and transferred togelatin-coated coverslips and allowed to attach. The stem cell spheresgradually settle to the floor surface, and come in contact with thegelatin coating. The spheres attach, flatten, and cells at the sphereboundaries that are in contact with gelatin change in morphology andbehavior over time. In particular, differentiation and proliferationoccur at the edges of the sphere where it is attached to the coatedsurface. Beyond these areas, the cells become motile and migrate awayfrom the sphere in all directions (FIG. 13).

Next, some of the Discospheres in culture were collected and plated ongelatin coated coverslips and allowed to attach and grow and werefollowed in real time for 3-4 days. (FIG. 14). The stem cell spheresattach to the coated surface, and attachment in the presence of serumcombined to cause the stem cell cluster to begin to flatten, and at thesame time differentiation and rapid proliferation occurs at the edges ofthe sphere where it was attached to the coated surface. Proliferationand/or migration away from the sphere and also differentiation of thecells occurred over time.

Discospheres were extracted from the Discosphere Culture Method, andplated on gelatin-coated coverslips in media containing 10% serum forapproximately eight hours (FIGS. 15-16) or three days (FIG. 17). Ateight hours, spheres were in primarily in a state between stemness(except the outer layers), but at the edges of the sphere, cells incontact with the gelatin-coated culture surface were undergoingdifferentiation. Vimentin was not expressed in the inner portion of theattached sphere where stem cells and early progenitors predominate (FIG.15). However, as the cells at the sphere edge attached to the gelatin,differentiation began to occur in association with migration andproliferation (see also FIG. 14). Vimentin was upregulated in disc cellsas they began to differentiate from stem cells or early progenitors atthe outer portion of the Discosphere.

At eight hours, CK8 was expressed in the inner portion of the attachedsphere (FIG. 16), and thus was associated with immature, stem-likephenotypy. Additionally, CK8 is a known marker of fetal nucleus pulposusand notochordal cells. Interestingly, the cellular cluster of stainingwas similar in its morphology and nature to that which was foundnaturally in fetal disc tissues. In fetal disc tissues, after earlydevelopment of the spine has occurred through instruction of theparaxial mesoderm by the notochord, notochordal cells persist asclusters or aggregates interspersed with nucleus pulposus cells. In thisscenario, the notochordal cells were positive for the expression of CK8,while the interspersed cells that make up the remainder of the disc werenegative.

However, at three days, CK8 is expressed at moderate levels in somecells, and weakly or not at all in others (especially cells plated assingle cells and not sphere clusters) (FIG. 17). Several cells do notexpress CK8, indicating that CK8 is likely down regulated in singlecells that are attached and likely differentiating. This indicates thatdifferentiation is associated with downregulation of CK-8. Thus, platingthe cells as single cells instead of as a cluster, and attaching thecells for a longer period of time, both downregulate CK8.

EXAMPLE 5 Cluster Culture Method

When the spheres from the discosphere culture method were mature, theywere then passaged in one of two manners. First, to expand the cellpopulation, they were passaged into the Cluster Culture Method bypreparing a single cell suspension using enzymatic digestion, andplating the cells in the Cluster Culture Method at a predefined platingdensity. This was an appropriate time to cryopreserve stem cells in asingle cell form. The cells were then grown in the Cluster CultureMethod in cycles to expand them as needed. The time it typically takesfor passaged cells from a single cell suspension to reach maturity ascluster like cell aggregates, was around 8-16 days. The end product ofcycling the cluster cells was a stem cell product of the culture method,which can be cryopreserved for use at a future date. The second approachto passage from the discosphere culture to the cluster culture was totransfer the disc stem cell spheres to the Cluster Culture directly. Thespheres grow into large clusters of heterogeneous morphology, reachingmaturity in 8-14 days. This was a stem cell product of the culturemethod, and the large cell clusters can be cryopreserved for future useas indicated (FIG. 18, reference numerals 1-3).

When the cells were confluent, the cluster culture was passaged bypreparing a single cell suspension using enzymatic digestion, and thecells were replated into the cluster culture method.

The Cluster Culture Method has as part of its media 10% serum,supplemented growth factors (Table 5), and uses tissue culture platesthat were coated so that the surfaces were ultra-low binding. Thus, thiswas a suspension culture. The cells were plated in low density andbecause they were in suspension, each cell grows into an aggregate-likecell cluster. However, because of the lack of methylcellulose, the cellswere free to move through the media and the clusters as well as smallercells compositions (even single cells) aggregated and grew into largeirregular clusters (FIG. 19). Aggregate cell clusters typically maturedin size after 8-12 days. Cell number and growth kinetic parameters fromthe Cluster Culture Method are presented in Table 6. The clusters can beprepared as a single cell suspension and replated back into clusterculture to expand the cell population.

TABLE 5 Key components of the Cluster Culture Method and N10 Media.COMPONENT AMOUNT/OTHER MEDIA DMEM/F12 SERUM 10% FGF 10 ng/ml EGF 10ng/ml CELL STATE SUSPENSION PLATING CELL DENSITY 1 × 10⁴ CELL SURFACEULTRA LOW BINDING

Protocol for 100 ml of N10 Media Concentrations of Reagents: 90 ml ofstock DMEM F12 Media 16.1 μg/ml of Putrescine 10 ml of Fetal Calf Serum(100 μM) 100 μl of stock 1000X Sodium Selenite 62.8 μg/ml ofProgesterone 100 μl of stock 1000X Insulin (20 μM) 100 μl of stock 1000XPutrescine 5.2 μg/ml of Sodium Selenite 100 μl of stock 1000XProgesterone (30 μM) 100 μl of stock 1000X Transferrin 50 μg/ml ofTransferrin. 5 μg/ml of Insulin

TABLE 6 Identification of important cell number and growth kineticparameters from the Cluster Culture Method. COMPONENT Number/Other CellState Suspension as Aggregate Clusters Time to Maturity (HealthyCellular Tissues) 10-16 days Time to Maturity (Degenerated AcellularTissues) 10-28 days

Cell clusters were extracted from the Cell Cluster Culture Method, andprepared as single cell suspensions by enzymatic digestion. The singlecells were plated on cover slips coated with gelatin in media containing1% serum for approximately five hours. CD133 was found to be highlyexpressed by most cells (FIG. 20) and emphasized the stem-like nature ofthe cell clusters generated in the Cell Cluster Method.

The disc stem cell clusters were collected and plated on gelatin coatedcoverslips and allowed to attach and grow. The stem cell clusters attachto the coated surface, and attachment plus serum combine to cause thestem cell cluster to begin to flatten, and at the same timedifferentiation and rapid proliferation occurs at the edges of thesphere where it was attached to the coated surface. Proliferation occursin concert with migration away from the sphere and also withdifferentiation of the cells (FIG. 21).

Cell clusters were extracted from the Cell Cluster Culture Method, andprepared as single cell suspensions by enzymatic digestion. The singlecells were plated on cover slips coated with Gelatin in chondrogenicdifferentiating media, and allowed to grow for 5 days. Collagen-2 alpha(FIG. 22A-C) and Vimentin (FIG. 22D-E) are known markers of mature disccells, and were expressed in nucleus pulposus cells. The morphology ofthese differentiated cells in tandem with their expression patterns wereconsistent with mature disc tissue derived cells.

EXAMPLE 6 Stem Cells Produced by the Discotek Culture Method Demonstratea Stem Cell Phenotype

OCT-4, in concert with NANOG and SOX-2, is a master transcriptionalregulator of embryonic stem cell biology. Its presence and activity in astem cell indicates an early pluripotent stem-like state. Thesetranscription factors regulate stem cell biology and early fetaldevelopment. For example, OCT-4 regulates clonal division and thesuppression of differentiation through their target genes, the so calledNOS genes (NANOG, OCT-4, AND SOX-2) (FIG. 23). A pOCT-4-eGFP reportertransgene was constructed in which all four response elements werecloned upstream of and regulating the expression of green fluorescentprotein (FIG. 24A). Disc stem cells were transfected with thepOCT-4-eGFP transgene, and stable transfectants were selected for theirresistance to the antibiotic G418 (via activation of the NEO resistancegene cassette also on the reporter plasmid) (FIG. 24B). The OCT-4promoter-GFP reporter transgene was activated in stably transfected disctissue stem cells in the various cultures described hereinabove,indicating that embryonic transcriptional machinery was active andinfluencing the pluripotency and stem-like behavior of disc tissue stemcells cultured in the Discotek Platform (for example, the Discosphereculture as shown in FIG. 25). The mechanism of these effects on stemcell biology that can regulated by OCT-4 occurs through a combination ofepigenetic modifications of the genome, and the transcriptionalregulation of key target genes important for embryonic stem cellphenotypy (e.g., NOS genes).

Disc stem cell spheres, when attached to a matrix coated surface, grewin a random manner without pattern or organization. However, if a narrowwindow of sphere concentration resulting in the achievement of specificinter-sphere distances, a self organized structure occurred that swirledaround the central attached sphere and formed boundaries withneighboring cell populations organized in a similar manner (FIG. 26).The result was a series of tissue culture units with definable anatomymade up of organized nucleus pulposus cells, and presumably organized bythe central stem cell cluster.

In disc cell cultures seeded from disc cell clusters, culture conditionsconverted cell growth from nonspecific to highly organized, and cellgrowth was contained within a certain radius around the attached discstem cell. Additionally, light microscopy and high magnificationmicroscopy using birefringent polarized light was used to furthercharacterize the appearance in culture of floating and attached cellfibers with a helical appearance. Finally, serial passaging and cellcounts from counterstained samples were used to estimate andcharacterize the growth potential of nucleus pulposus cells in extendeddisc stem cell and disc cell cultures.

As described hereinabove, collagen like structures of various sizes andconformations were found to spontaneously appear in both disc stem celland disc cell cultures. The fibers were elongated and relativelystraight structures and a significant proportion had a helicalconformation that could be observed at very high magnification. Stemcell clusters and nucleus pulposus cells were found to attach, migratealong, grow upon, and grow from the fibers. The amount of fibersobserved increased the longer the cells were in culture. Further, whendisc stem cells were plated on ECM coated surfaces at specific cellsurface plating densities, the cell progeny were found to emanate fromthem in a very specific, reproducible, and organized cell pattern orarray. These arrays consisted of cells migrating from and proliferatingaround the original cluster in a circular manner with one group of cellslayered along another. Finally, these cultures were passaged severaltimes and the cell yield and surface area covered was calculated andfound to be exponentially greater than that of the original seeded stemcell clusters.

The natural inclination of adult human disc stem cells to form fibers,produce progeny that grow in an organized manner, and give rise to largenumbers of nucleus pulposus cells demonstrates significant potential forstem cell based tissue engineering to treat degenerative disc disease.

EXAMPLE 7 Isolation and Characterization of Disc Stem Cells from HumanAdult Disc Tissue

Degenerative disc disease (DDD) is a chronic degeneration of disc tissuethat occurs with aging, and the underlying pathologic process for mostspinal joint disease. Isolation and characterization of adult disc stemcells from healthy disc tissue is a step forward for developing stemcell based therapeutic approaches to treat DDD. The aim of this studywas to determine whether adult disc stem cells could be isolated fromhealthy and degenerated adult human disc tissue isolated from humanpatients undergoing disc surgery. Using the novel disc stem cell cultureplatform described hereinabove, disc stem cells were cultured forexpansion and subjected to stem cell assays including clonal growthanalysis and differentiation studies. Immunohistochemistry was used todemonstrate the expression of biomarkers of mature nucleus pulposuscells. Basic tissue engineering methods were used to characterize thegrowth kinetics and the total cell yield of nucleus pulposus cellcultures derived from disc stem cells in vitro.

Adult disc stem cells were efficiently and reproducibly isolated frompatient specimens that were relatively healthy in nature. Disc stemcells grew clonally from single cells into stem cell clusters that werespherical. Passaging stem cells as a single cell suspension by replatingthem in the same culture system for expansion resulted in a lineargrowth rate. When disc stem cells were attached to extracellular matrixcoated surfaces in the presence of serum, they rapidly differentiatedinto mature nucleus pulposus cells as verified by morphology, cellbiology assays, and the expression of collagen 2, aggrecan, andvimentin. Additionally, nucleus pulposus cell progeny grew for severalpassages and could be expanded exponentially when attached to a matrixcoated culture surface in the presence of chondrogenic media. Thisreport demonstrates the ability to isolate, culture, and study in vitroadult disc stem cells from healthy adult disc tissue. Disc stem cellsreadily give rise to nucleus pulposus progeny with significant growthpotential.

Adult disc stem cells were efficiently and reproducibly isolated fromdegenerate patient specimens. Disc stem cells grew clonally into stemcell clusters. Stem cell and disc cell growth in culture was assessedand although expansion was possible, there was significant variabilityin the results. Passaging the stem cell clusters as single cellsuspensions by replating them in the same culture system for expansionresulted in a linear growth rate in some patients but not others. Discstem cells, when attached to extracellular matrix coated surfaces in thepresence of serum, rapidly differentiated into mature nucleus pulposuscells in all patient samples as verified by morphology, cell biologyassays, and the expression of collagen 2, aggrecan, and vimentin.Additionally, nucleus pulposus cell progeny that grew for severalpassages and could be expanded exponentially in 60% of all patientsamples when grown in chondrogenic tissue culture conditions. We havedemonstrated the ability to isolate, culture, and study in vitro adultdisc stem cells from degenerated disc tissue. All patient's stem cellsdemonstrated significant growth potential when cultured in chondrogenicconditions. The observation of intra-patient variability likely relatesto a combination of patient age, and disease status. The identificationof biomarkers for stem cell viability in degenerated disc tissues fromhuman adult patients is an important step for securing this patientpopulation as a donor source for stem cell based therapeutics.

EXAMPLE 8 In Vitro Tissue Engineering of Nucleus Pulposus Using AdultDisc Stem Cells

Adult disc stem cell potential when combined with tissue engineeringprinciples has significant potential for the treatment of DDD. The aimof this study was to investigate the in vitro tissue engineeringpotential of adult disc stem cells derived from healthy adult disctissue. The ability of disc stem cells to generate functional nucleuspulposus cells and fabricate tissue in vitro was assessed.

Study design: We successfully isolated disc stem cells from healthy disctissue for further study. Tissue engineering was used to characterizethe rate of cell growth and several cell biology parameters. Novel 3dimensional (D) tissue engineering models were created to characterizethe ability of disc stem cells to grow in scaffolds, fill defects, andsecrete extracellular matrix (ECM).

Methods: disc stem cells were plated in basic tissue engineering assaysand followed over time with light microscopy. The rate of development ofprogeny from the stem cells was characterized, as well as their abilityto migrate, the velocity of motility, differentiation events, and thedegree of proliferation. Subsequently, a novel 3 D tissue engineeringmodel was used to determine the ability of adult disc stem cells to fillspatial defects that are seeded with scaffolds allowing cell migrationin all directions. The ability of disc stem cells and seeded scaffoldsto fabricate tissue and lay down ECM was determined with histologicstains and light microscopy. In both models, histology andimmunohistochemistry for disc biomarkers was used to verify and furthercharacterize nucleus pulposus cell progeny.

Results: disc stem cells readily attached to ECM coated culture plates.Progenitor-like cells in contact with the coated surface migrated fromthe outside in, and spread across the available culture area in 96hours. Migration was followed by proliferation, and the combinationresulted in large surface areas surrounding the stem cell cluster beingcovered with nucleus pulposus cells. Using a 3 D scaffold based tissueengineering assay, disc stem cells were able seed scaffolds effectively,migrate multidirectionally, and proliferate to fill structural defects.Post-culture gross inspection revealed a disc like structure resemblingnucleus pulposus. The neoengineered tissue was sectioned and histologicanalysis revealed tissue fabrication histology that was moderately celldense with extensive ECM. Stem cell cluster remnants were easilylocated. A variation of this model was used to observe in real time themorphology and migration of disc stem cells as they emerged from discstem cell clusters. We have characterized the ability of disc stem cellsto effectively populate tissue engineering assays in a relevant andfunctional manner.

EXAMPLE 9 Disc with Endplate Tissue Scaffold Model

The aim of this study was to investigate the potential of human adultdisc stem cells to create nucleus pulposus tissue in a denucleatedrabbit disc annulus with endplates that serves as an effective 3dimensional (D) scaffold. Denucleated rabbit annulus with thin bonyendplates was prepared and used as a scaffold into which disc stem cellswere injected to assess their ability to grow into disc tissue in vitro.Rabbit discs with bony endplates intact were dissected out as units fromthe lumbar portion of the spine. The discs were treated biochemicallyand mechanically to denucleate them from any central disc tissue,leaving only the annulus, inner matrix protein scaffolding, and the bonyendplates. Disc stem cells were introduced and the disc units werecultured in chondrogenic media in a tissue culture incubator. A dynamiccompressive force was applied to the disc unit on a daily basis, twiceper day. The tissue engineering experiment was followed for 3 months,and the discs were then sectioned and assayed. Histologic stains forbone, fat, and cartilage were used to characterize the tissue. Theexpression of the extracellular matrix proteins collagen II and collagenI, and the proliferation marker, KI67, were assayed usingimmunohistochemistry.

Results: Post-culture gross inspection revealed the disc unit had a fullbody that had physical properties such as tensile strength andresistance to compressive forces. The neoengineered tissue was sectionedand histologic analysis revealed a healthy appearing cartilage liketissue and no evidence of bone or fat formation. Expression of collagenII was significant, and the expression of collagen 1 was minimal,consistent with healthy disc tissue. Importantly, no KI67 expressioncould be detected, indicating that disc stem cells and their progeny hadceased to proliferate and likely differentiated.

What is claimed is:
 1. A method of amplifying and enriching a disc stemcell population comprising the steps of: a. suspending a sphere-likecluster of disc stem cells or an isolated disc stem cell from saidsphere-like cell cluster in a medium comprising 6-10% serum, basicfibroblast growth factor (bFGF), and epidermal growth factor (EGF) andlacking methylcellulose; b. distributing the suspension onto ultra-lowbinding culture plates; and c. culturing said suspension for 8-16 daysto produce a mature sphere of heterogeneous morphology having a diameterof roughly 100-600 microns, thereby amplifying and enriching a disc stemcell population.
 2. The method of claim 1, wherein said disc stem cellpopulation is a mammalian disc stem cell population.
 3. The method ofclaim 1, wherein said disc stem cell population is a human disc stemcell population.
 4. The method of claim 1, wherein said disc stem cellpopulation is isolated from degenerated disc tissue.
 5. The method ofclaim 1, wherein said disc stem cell population is isolated from healthydisc tissue.
 6. The method of claim 5, wherein said healthy disc tissueis fetal, neonatal, or young adult tissue.
 7. The method of claim 1,wherein said serum is present at a concentration of 10%.
 8. The methodof claim 1, wherein said bFGF is present at a concentration of 10 ng/ml.9. The method of claim 1, wherein said EGF is present at a concentrationof 10 ng/ml.
 10. The method of claim 1, wherein said serum is fetal calfserum (FCS).
 11. The method of claim 1, wherein said medium is DMEM/F12,N10, or a combination thereof.
 12. The method of claim 1, wherein saidmedium further comprises putrescine, progesterone, sodium selenite,transferrin, and insulin.
 13. The method of claim 1, wherein saidsuspension is distributed at a concentration of 1×10⁵ cells/ml.
 14. Themethod of claim 1, further comprising the step of producing a singlecell suspension from a sphere-like disc stem cell cluster usingenzymatic digestion prior to step (a).
 15. The method of claim 1,further comprising incubating at least a portion of said disc stem cellpopulation in a chondrogenic differentiating media, wherein at least aportion of said disc stem cell population expresses markers of maturedisc cells after incubation with said chondrogenic differentiatingmedia.
 16. The method of claim 15, wherein said markers comprisecollagen-2 alpha, vimentin, or a combination thereof.
 17. The method ofclaim 1, further comprising the step of cryopreserving said maturesphere of heterogeneous morphology.
 18. A method of inhibiting, ordecreasing the likelihood of damage to or disease of acartilage-containing joint of a subject, comprising; a. suspending asphere-like cluster of disc stem cells or an isolated disc stem cellfrom said sphere-like cell cluster in a medium comprising 6-10% serum,basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)and lacking methylcellulose; b. distributing the suspension ontoutra-low binding culture plates; c. culturing said suspension for 8-16days to produce a mature sphere of heterogeneous morphology having adiameter of roughly 100-600 microns; and d. administering to saidsubject said mature sphere of heterogeneous morphology therebyinhibiting, or decreasing the likelihood of damage to or disease of thecartilage-containing joint of said subject.
 19. The method of claim 18,wherein the administration is percutaneous.
 20. The method of claim 18,wherein the administration is directly to the joint of said subject aspart of a surgical procedure.
 21. The method of claim 18, wherein saidjoint is a hip joint.
 22. The method of claim 18, wherein said joint isa knee joint.
 23. The method of claim 18, wherein said joint is a spinaljoint.
 24. A method of amplifying and enriching a disc stem cellpopulation comprising the steps of: a. suspending a sphere-like clusterof disc stem cells or an isolated disc stem cell from said sphere-likecell cluster in a medium comprising 6-10% serum, basic fibroblast growthfactor (bFGF), and epidermal growth factor (EGF) and lackingmethylcellulose; b. distributing the suspension onto ultra-low bindingculture plates; and c. culturing said suspension for 8-16 days toproduce a mature sphere of heterogeneous morphology, wherein the numberof cells in the mature sphere is approximately 100-500 cells, therebyamplifying and enriching a disc stem cell population.
 25. The method ofclaim 24, wherein said disc stem cell population is a mammalian discstem cell population.
 26. The method of claim 24, wherein said disc stemcell population is a human disc stem cell population.
 27. The method ofclaim 24, wherein said suspension is distributed at a concentration of1×10⁵ cells/ml.
 28. The method of claim 24, wherein said EGF is presentat a concentration of 10 ng/ml.
 29. A method of inhibiting, ordecreasing the likelihood of damage to or disease of acartilage-containing joint of a subject, comprising; a. suspending asphere-like cluster of disc stem cells or an isolated disc stem cellfrom said sphere-like cell cluster in a medium comprising 6-10% serum,basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF)and lacking methylcellulose; b. distributing the suspension ontoultra-low binding culture plates; c. culturing said suspension for 8-16days to produce a mature sphere of heterogeneous morphology wherein thenumber of cells in the mature sphere is approximately 100-500 cells; andd. administering to said subject said mature sphere of heterogeneousmorphology thereby inhibiting, or decreasing the likelihood of damage toor disease of the cartilage-containing joint of said subject.
 30. Themethod of claim 29, wherein the administration is percutaneous.
 31. Themethod of claim 29, wherein the administration is directly to the jointof said subject as part of a surgical procedure.
 32. The method of claim18, wherein said joint is a hip joint.
 33. The method of claim 18,wherein said joint is a knee joint.
 34. The method of claim 18, whereinsaid joint is a spinal joint.